Electron transport proteins

ABSTRACT

The invention provides human electron transport proteins (NHETP) and polynucleotides which identify and encode NHETP. The invention also provides expression vectors, host cells, agonists, antibodies and antagonists. The invention also provides methods for treating or preventing disorders associated with expression of NHETP.

FIELD OF THE INVENTION

This invention relates to nucleic acid and amino acid sequences ofelectron transport proteins and to the use of these sequences in thediagnosis, prevention, and treatment of cancer, immune disorders, andreproductive disorders.

BACKGROUND OF THE INVENTION

Electron transport is the general process in cells by which electronsgenerated from the oxidation of molecules such as NADH and FADH₂ aretransferred, through the action of various enzymes, to a series ofelectron carriers. These electron carriers may act as electron donorsthemselves for various reductive reactions in the cell or may transporttheir electrons to other electron carriers along an electron transportchain. The change in oxidation potential as electrons are passed alongsuch a chain generates energy which may be used by the cell.

The mitochondrial electron transport (or respiratory) chain is a seriesof enzyme complexes in the mitochondrial membrane responsible for thetransport of electrons from NADH through a series of redox centers(electron carriers) within these complexes to oxygen and for thecoupling of this oxidation to the synthesis of ATP (oxidativephosphorylation). ATP provides the primary source of energy for drivinga cell's many energy-requiring reactions.

Most electron carriers are prosthetic groups, such as flavins, heme,iron-sulfur clusters and copper, bound to protein particles. Ubiquinone(Coenzyme Q) is the only electron carrier that is not protein bound. Thecytochromes (Cyts) are one type of electron carrier protein; cytochromesare related to one another by the presence of a bound heme groupconsisting of a porphyrin ring containing a tightly bound iron atom. Theiron atom serves as the actual electron carrier by changing from theferric to the ferrous state when accepting an electron. Iron-sulfurproteins are a second major family of electron carriers in which eithertwo or four iron atoms are bound to sulfur atoms and to cysteine sidechains forming an iron-sulfur center. Ubiquinone, the simplest ofelectron carriers, includes a quinone ring attached to a hydrophobictail which anchors it to the mitochondrial membrane. In addition to sixdifferent heme-linked cytochromes, more than six iron-sulfur centers,and ubiquinone, there are also two copper atoms and a flavin (FMN)serving as electron carriers in the pathway from NADH to oxygen.

The key enzyme complexes in the respiratory chain are NADH:ubiquinoneoxidoreductase (NADH-D), succinate:ubiquinone oxidoreductase, cytochromec₁ -b oxidoreductase, cytochrome c oxidase (COX), and ATP synthase. Allof these complexes are located on the inner matrix side of themitochondrial membrane except succinate:ubiquinone oxidoreductase, whichis located on the cytosolic side. NADH-D accomplishes the first step inthe respiratory chain by accepting electrons from NADH and passing themthrough a flavin molecule and several iron-sulfur centers to ubiquinone.Succinate:ubiquinone oxidoreductase also transports electrons generatedby oxidation of succinate to fumarate in the citric acid cycle throughelectron carriers (FAD and iron-sulfur centers) to the membrane boundubiquinone. Cytochrome c₁ -boxidoreductase accepts electrons fromubiquinone and passes them on to cytochrome c. COX accepts electronsfrom cytochrome c and catalyzes the last, and most important, transferof electrons to oxygen. Energy released in the course of each of theseelectron transfers is harnessed by ATP synthase to form ATP (oxidativephosphorylation).

NADH-D, the largest of these complexes with an estimated mass of 800kDa, contains some 40 polypeptide subunits of widely varying size andcomposition. The polypeptide composition of NADH-D is similar in avariety of mammalian species including rat, rabbit, cow, and human(Cleeter, M. W. J. and Ragan, C. I. (1985) Biochem. J. 230: 739-46). Thebest characterized NADH-D is from bovine heart mitochondria and iscomposed of 41 polypeptides (Walker, J. E. et al. (1992) J. Mol. Biol.226: 1051-72). Seven of these polypeptides are encoded by mitochondrialDNA, while the remaining 34 are nuclear gene products that are importedinto the mitochondria. Six of these imported polypeptides arecharacterized by N-terminal signal peptide sequences which target thesepolypeptides to the mitochondria and are then cleaved from the matureproteins. A second group of polypeptides lack N-terminal targetingsequences and appear to contain import signals which lie within themature protein (Walker et al., supra). The measured molecular masses ofseveral of the smaller polypeptides, B8, B13, B14, B15, and B22, areconsistent with post-translational removal of the terminal methionineresidue and N-acetylation of the adjacent amino acid.

The functions of many of the individual subunits in NADH-D are largelyunknown. The 24-, 51 -, and 75-kDa subunits have been identified asbeing catalytically important in electron transport, with the 51 -kDasubunit forming part of the NADH binding site and containing the flavinmoiety that is the initial electron acceptor (Ali, S. T. et al. (1993)Genomics 18:435-39). The location of other functionally importantgroups, such as the electron-carrying iron-sulfate centers, remains tobe determined. Many of the ;maller subunits (<30 kDa) containhydrophobic sequences that may be folded into membrane spanning(α-helices. These subunits presumably are anchored into the innermembrane of the mitochondria and interact via more hydrophilic parts oftheir sequence with globular proteins in the large extrinsic domain ofNADH-D.

COX is composed of thirteen polypeptide subunits, three of which aremitochondrial gene products, and the ten remaining subunits of which arenuclear gene products (Lomax, M. I. et al. (1990) Gene 86: 209-16). Thecatalytic and protein-transducing functions and the site of interactionwith cytochrome c are all associated with the mitochondrial geneproducts, subunits 1 through 3. The exact functions of the ten smaller,nuclear-encoded subunits, 4 through 13, are unknown, but it has beensuggested that they regulate oxidative energy output (Lomax et al.,supra).

NADH cytochrome b5 reductase is an enzyme that specifically serves tooxidize the electron carrier cytochrome b5 which, in turn, becomes acentral electron donor for various reductive reactions occurring on thecytoplasmic surface of liver endoplasmic reticulum (Strittmatter, P. etal. (1992) J. Biol. Chem. 267: 2519-23). Both cytochrome b5 reductase,and cytochrome b5 are amphipathic molecules composed of globularhydrophilic catalytic domains linked through short flexible sequences tomembrane-anchoring hydrophobic domains that serve to orient thecatalytic sites at the membrane-aqueous interface and permit rapidelectron transfer. Three lysine residues in cytochrome b5 reductase,K41, K125, and K163, are implicated in the formation of charged ionpairs with carboxyl groups on cytochrome b5 during interactions betweenthe active sites of the two proteins (Strittmatter, P. et al. (1990) J.Biol. Chem. 265: 21709-13). Site-directed mutagenesis studiesdemonstrate marked decreases in catalytic efficiency when any of thesethree lysine residues are replaced by negatively charged amino acids(Strittmatter et al. (1992), supra).

Defects and altered expression of NADH-D are associated with a varietyof human diseases, including neurodegenerative diseases, myopathies, andcancer (Singer, T. P. et al. (1995) Biochim. Biophys. Acta 1271:211-19;Selvanayagam, P. and Rajaraman, S. (1996) Lab. Invest. 74:592-99). Inaddition, NADH-D reduction of the quinone moiety in chemotherapeuticagents such as doxorubicin is believed to contribute to the antitumoractivity and/or mutagenicity of these drugs (Akman, S. A. et al. (1992)Biochemistry 31:3500-6).

The discovery of new electron transport proteins and the polynucleotidesencoding them satisfies a need in the art by providing new compositionswhich are useful in the diagnosis, prevention, and treatment of cancer,immune disorders, and reproductive disorders.

SUMMARY OF THE INVENTION

The invention features three substantially purified polypeptides,electron transport proteins NHETP-1, NHETP-2, and NHETP-3 (referred tocollectively as NHETP), having the amino acid sequences shown in SEQ IDNO:1, SEQ ID NO:3, and SEQ ID NO:5 or fragments thereof.

The invention further provides an isolated and substantially purifiedpolynucleotide sequence encoding the polypeptide comprising the aminoacid sequence of SEQ ID NO:1 or fragments thereof and a compositioncomprising said polynucleotide sequence. The invention also provides apolynucleotide sequence which hybridizes under stringent conditions tothe polynucleotide sequence encoding the amino acid sequence SEQ IDNO:1, or fragments of said polynucleotide sequence. The inventionfurther provides a polynucleotide sequence comprising the complement ofthe polynucleotide sequence encoding the amino acid sequence of SEQ IDNO:1, or fragments or variants of said polynucleotide sequence.

The invention also provides an isolated and purified sequence comprisingSEQ ID NO:2 or variants thereof. In addition, the invention provides apolynucleotide sequence which hybridizes under stringent conditions tothe polynucleotide sequence of SEQ ID NO:2. the invention also providesa polynucleotide sequence comprising the complement of SEQ ID NO:2, orfragments or variants thereof.

The present invention further provides an expression vector containingat least a fragment of any of the claimed polynucleotide sequences. Inyet another aspect. the expression vector containing the polynucleotidesequence is contained within a host cell.

The invention also provides a method for producing a polypeptidecomprising the amino acid sequence of SEQ ID NO:1, or a fragmentthereof, the method comprising the steps of: a) culturing the host cellcontaining an expression vector containing at least a fragment of thepolynucleotide sequence encoding NHETP-1 under conditions suitable forthe expression of the polypeptide; and b) recovering the polypeptidefrom the host cell culture.

The invention also provides a pharmaceutical composition comprising atsubstantially purified NHETP-1 having the amino acid sequence of SEQ IDNO:1 in conjunction with a suitable pharmaceutical carrier.

The invention also provides a purified antagonist of the polypeptide ofSEQ ID NO:1. In one aspect the invention provides a purified antibodywhich binds to a polypeptide comprising the amino acid sequence of SEQID NO:1.

Still further, the invention provides a purified agonist of thepolypeptide of SEQ ID NO:1.

The invention also provides a method for treating or preventing cancercomprising administering to a subject in need of such treatment aneffective amount of a purified antagonist of NHETP-1.

The invention also provides a method for treating or preventing animmune disorder comprising administering to a subject in need of suchtreatment an effective amount of a purified antagonist of NHETP-1.

The invention also provides a method for treating or preventing areproductive disorder comprising administering to a subject in need ofsuch treatment an effective amount of a purified antagonist of NHETP-1.

The invention also provides a method for detecting a polynucleotidewhich encodes NHETP-1 in a biological sample comprising the steps of: a)hybridizing the complement of the polynucleotide sequence which encodesSEQ ID NO:1 to nucleic acid material of a biological sample, therebyforming a hybridization complex; and b) detecting the hybridizationcomplex, wherein the presence of the complex correlates with thepresence of a polynucleotide encoding NHETP-1 in the biological sample.In one aspect the nucleic acid material of the biological sample isamplified by the polymerase chain reaction prior to hybridization.

The invention further provides an isolated and substantially purifiedpolynucleotide sequence encoding the polypeptide comprising the aminoacid sequence of SEQ ID NO:3 or fragments thereof and a compositioncomprising said polynucleotide sequence. The invention also provides apolynucleotide sequence which hybridizes under stringent conditions tothe polynucleotide sequence encoding the amino acid sequence SEQ IDNO:3, or fragments of said polynucleotide sequence. The inventionfurther provides a polynucleotide sequence comprising the complement ofthe polynucleotide sequence encoding the amino acid sequence of SEQ IDNO:3, or fragments or variants of said polynucleotide sequence.

The invention also provides an isolated and purified sequence comprisingSEQ ID NO:4 or variants thereof. In addition, the invention provides apolynucleotide sequence which hybridizes under stringent conditions tothe polynucleotide sequence of SEQ ID NO:4. The invention also providesa polynucleotide sequence comprising the complement of SEQ ID NO:4, orfragments or variants thereof.

The present invention further provides an expression vector containingat least a fragment of any of the claimed polynucleotide sequences. Inyet another aspect, the expression vector containing the polynucleotidesequence is contained within a host cell.

The invention also provides a method for producing a polypeptidecomprising the amino acid sequence of SEQ ID NO:3, or a fragmentthereof, the method comprising the steps of: a) culturing the host cellcontaining an expression vector containing at least a fragment of thepolynucleotide sequence encoding NHETP-2 under conditions suitable forthe expression of the polypeptide; and b) recovering the polypeptidefrom the host cell culture.

The invention also provides a pharmaceutical composition comprising asubstantially purified NHETP-2 having the amino acid sequence of SEQ IDNO:3 in conjunction with a suitable pharmaceutical carrier.

The invention also provides a purified antagonist of the polypeptide ofSEQ ID NO:3. In one aspect the invention provides a purified antibodywhich binds to a polypeptide comprising the amino acid sequence of SEQID NO:3.

Still further, the invention provides a purified agonist of thepolypeptide of SEQ ID NO:3.

The invention also provides a method for treating or preventing cancercomprising administering to a subject in need of such treatment aneffective amount of a purified antagonist of NHETP-2.

The invention also provides a method for treating or preventing animmune disorder comprising administering to a subject in need of suchtreatment an effective amount of a purified antagonist of NHETP-2.

The invention also provides a method for detecting a polynucleotidewhich encodes NHETP-2 in a biological sample comprising the steps of: a)hybridizing the complement of the polynucleotide sequence which encodesSEQ ID NO:3 to nucleic acid material of a biological sample, therebyforming a hybridization complex; and b) detecting the hybridizationcomplex, wherein the presence of the complex correlates with thepresence of a polynucleotide encoding NHETP-2 in the biological sample.In one aspect the nucleic acid material of the biological sample isamplified by the polymerase chain reaction prior to hybridization.

The invention further provides an isolated and substantially purifiedpolynucleotide sequence encoding the polypeptide comprising the aminoacid sequence of SEQ ID NO:5 or fragments thereof and a compositioncomprising said polynucleotide sequence. The invention also provides apolynucleotide sequence which hybridizes under stringent conditions tothe polynucleotide sequence encoding the amino acid sequence SEQ IDNO:5, or fragments of said polynucleotide sequence. The inventionfurther provides a polynucleotide sequence comprising the complement ofthe polynucleotide sequence encoding the amino acid sequence of SEQ IDNO:5, or fragments or variants of said polynucleotide sequence.

The invention also provides an isolated and purified sequence comprisingSEQ ID NO:6 or variants thereof. In addition, the invention provides apolynucleotide sequence which hybridizes under stringent conditions tothe polynucleotide sequence of SEQ ID NO:6. The invention also providesa polynucleotide sequence comprising the complement of SEQ ID NO:6, orfragments or variants thereof.

The present invention further provides an expression vector containingat least a fragment of any of the claimed polynucleotide sequences. Inyet another aspect, the expression vector containing the polynucleotidesequence is contained within a host cell.

The invention also provides a method for producing a polypeptidecomprising the amino acid sequence of SEQ ID NO:5, or a fragmentthereof, the method comprising the steps of: a) culturing the host cellcontaining an expression vector containing at least a fragment of thepolynucleotide sequence encoding NHETP-3 under conditions suitable forthe expression of the polypeptide; and b) recovering the polypeptidefrom the host cell culture.

The invention also provides a pharmaceutical composition comprising asubstantially purified NHETP-3 having the amino acid sequence of SEQ IDNO:5 in conjunction with a suitable pharmaceutical carrier.

The invention also provides a purified antagonist of the polypeptide ofSEQ ID NO:5. In one aspect the invention provides a purified antibodywhich binds to a polypeptide comprising the amino acid sequence of SEQID NO:5.

Still further, the invention provides a purified agonist of thepolypeptide of SEQ ID NO:5.

The invention also provides a method for treating or preventing cancercomprising administering to a subject in need of such treatment aneffective amount of a purified antagonist of NHETP-3.

The invention also provides a method for treating or preventing animmune disorder comprising administering to a subject in need of suchtreatment an effective amount of a purified antagonist of NHETP-3.

The invention also provides a method for treating or preventing areproductive disorder comprising administering to a subject in need ofsuch treatment an effective amount of a purified antagonist of NHETP-3.

The invention also provides a method for detecting a polynucleotidewhich encodes NHETP-3 in a biological sample comprising the steps of: a)hybridizing the complement of the polynucleotide sequence which encodesSEQ ID NO:5 to nucleic acid material of a biological sample, therebyforming a hybridization complex; and b) detecting the hybridizationcomplex, wherein the presence of the complex correlates with thepresence of a polynucleotide encoding NHETP-3 in the biological sample.In one aspect the nucleic acid material of the biological sample isamplified by the polymerase chain reaction prior to hybridization.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B, 1C, and 1D show the amino acid sequence (SEQ ID NO:1) andnucleic acid sequence (SEQ ID NO:2) of NHETP-1. The alignment wasproduced using MACDNASIS PRO software (Hitachi Software Engineering Co.Ltd. San Bruno, Calif.).

FIGS. 2A and 2B show the amino acid sequence (SEQ ID NO:3) and nucleicacid sequence (SEQ ID NO:4) of NHETP-2. The alignment was produced usingMACDNASIS PRO software.

FIGS. 3A and 3B shows the amino acid sequence (SEQ ID NO:5) and nucleicacid sequence (SEQ ID NO:6) of NHETP-3. The alignment was produced usingMACDNASIS PRO software.

FIG. 4 shows the amino acid sequence alignments between NHETP-1 (SEQ IDNO:1), and cytochrome b5 reductase from cow, b5R (GI 162941; SEQ IDNO:7), produced using the multisequence alignment program of DNASTARsoftware (DNASTAR Inc, Madison Wis.).

FIG. 5 shows the amino acid sequence alignments between NHETP-2 (SEQ IDNO:3), and cytochrome coxidase subunit 4 from human, COX4 (GI 180935;SEQ ID NO:8), produced using the multisequence alignment program ofDNASTAR software.

FIG. 6 shows the amino acid sequence alignments between NHETP-3 (SEQ IDNO:5), and NADH dehydrogenase subunit B14 from cow (GI 240; SEQ IDNO:9), produced using the multisequence alignment program of DNASTARsoftware.

FIGS. 7A and 7B show the hydrophobicity plots for NHETP-1 (SEQ IID NO:1)and bovine, b5R (SEQ ID NO:7), respectively; the positive X axisreflects amino acid position, and the negative Y axis, hydrophobicity(MACDNASIS PRO software).

FIGS. 8A and 8B show the hydrophobicity plots for NHETP-2 (SEQ ID NO:3)and human COX4 (SEQ ID NO:8), respectively; the positive X axis reflectsamino acid position, and the negative Y axis, hydrophobicity (MACDNASISPRO software).

FIGS. 9A and 9B show the hydrophobicity plots for NHETP-3 (SEQ ID NO:5)and NADH dehydrogenase subunit B14 from cow (SEQ ID NO:9), respectively;the positive X axis reflects amino acid position, and the negative Yaxis, hydrophobicity (MACDNASIS PRO software).

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms "a", "an", and "the" include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to "ahost cell" includes a plurality of such host cells, reference to the"antibody" is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications mentionedherein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

DEFINITIONS

NHETP, as used herein, refers to the amino acid sequences ofsubstantially purified NHETP obtained from any species, particularlymammalian, including bovine, povine, porcine, murine, equine, andpreferably human, from any source whether natural, synthetic,semi-synthetic, or recombinant.

The term "agonist", as used herein, refers to a molecule which, whenbound to NHETP, increases or prolongs the duration of the effect ofNHETP. Agonists may include proteins, nucleic acids, carbohydrates, orany other molecules which bind to and modulate the effect of NHETP.

An "allele" or "allelic sequence", as used herein, is an alternativeform of the gene encoding NHETP. Alleles may result from at least onemutation in the nucleic acid sequence and may result in altered mRNAs orpolypeptides whose structure or function may or may not be altered. Anygiven natural or recombinant gene may have none, one, or many allelicforms. Common mutational changes which give rise to alleles aregenerally ascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

"Altered" nucleic acid sequences encoding NHETP as used herein includethose with deletions, insertions, or substitutions of differentnucleotides resulting in a polynucleotide that encodes the same or afunctionally equivalent NHETP. Included within this definition arepolymorphisms which may or may not be readily detectable using aparticular oligonucleotide probe of the polynucleotide encoding NHETP,and improper or unexpected hybridization to alleles, with a locus otherthan the normal chromosomal locus for the polynucleotide sequenceencoding NHETP. The encoded protein may also be "altered" and containdeletions, insertions, or substitutions of amino acid residues whichproduce a silent change and result in a functionally equivalent NHETP.Deliberate amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, an/or the amphipathic nature of the residues as long asthe biological or immunological activity of NHETP is retained. Forexample, negatively charged amino acids may include aspartic acid andglutamic acid; positively charged amino acids may include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values may include leucine, isoleucine, andvaline, glycine and alanine, asparagine and glutamine, serine andthreonine, and phenylalanine and tyrosine.

"Amino acid sequence" as used herein refers to an oligopeptide, peptide,polypeptide, or protein sequence, and fragment thereof, and to naturallyoccurring or synthetic molecules. Fragments of NHETP are preferablyabout 5 to about 15 amino acids in length and retain the biologicalactivity or the immunological activity of NHETP. Where "amino acidsequence" is recited herein to refer to an amino acid sequence of anaturally occurring protein molecule, amino acid sequence, and liketerms, are not meant to limit the amino acid sequence to the complete,native amino acid sequence associated with the recited protein molecule.

"Amplification" as used herein refers to the production of additionalcopies of a nucleic acid sequence and is generally carried out usingpolymerase chain reaction (PCR) technologies well known in the art(Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, a LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y.).

The term "antagonist" as used herein, refers to a molecule which, whenbound to NHETP, decreases the amount or the duration of the effect ofthe biological or immunological activity of NHETP. Antagonists mayinclude proteins, nucleic acids, carbohydrates, antibodies or any othermolecules which decrease the effect of NHETP.

As used herein, the term "antibody" refers to intact molecules as wellas fragments thereof, such as Fab, F(ab')₂, and Fv, which are capable ofbinding the epitopic' determinant. Antibodies that bind NHETPpolypeptides can be prepared using intact polypeptides or fragmentscontaining small peptides of interest as the immunizing antigen. Thepolypeptide or oligopeptide used to immunize an animal can be derivedfrom the translation of RNA or synthesized chemically and can beconjugated to a carrier protein, if desired. Commonly used carriers thatare chemically coupled to peptides include bovine serum albumin andthyroglobulin, keyhole limpet hemocyanin. The coupled peptide is thenused to immunize the animal (e.g., a mouse, a rat, or a rabbit).

The term "antigenic determinant", as used herein, refers to thatfragment of a molecule (i.e., an epitope) that makes contact with aparticular antibody. When a protein or fragment of a protein is used toimmunize a host animal, numerous regions of the protein may induce theproduction of antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

The term "antisense", as used herein, refers to any compositioncontaining nucleotide sequences which are complementary to a specificDNA or RNA sequence. The term "antisense strand" is used in reference toa nucleic acid strand that is complementary to the "sense" strand.Antisense molecules include peptide nucleic acids and may be produced byany method including synthesis or transcription. Once introduced into acell, the complementary nucleotides combine with natural sequencesproduced by the cell to form duplexes and block either transcription ortranslation. The designation "negative" is sometimes used in referenceto the antisense strand, and "positive" is sometimes used in referenceto the sense strand.

The term "biologically active", as used herein, refers to a proteinhaving structural, regulatory, or biochemical functions of a naturallyoccurring molecule. Likewise, "immunologically active" refers to thecapability of the natural, recombinant, or synthetic NHETP, or anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

The terms "complementary" or "complementarity", as used herein, refer tothe natural binding of polynucleotides under permissive salt andtemperature conditions by base-pairing. For example, the sequence"A-G-T" binds to the complementary sequence "T-C-A".

Complementarity between two single-stranded molecules may be "partial",in which only some of the nucleic acids bind, or it may be complete whentotal complementaiity exists between the single stranded molecules. Thedegree of complementarity between nucleic acid strands has significanteffects on the efficiency and strength of hybridization between nucleicacid strands. This is of particular importance in amplificationreactions, which depend upon binding between nucleic acids strands andin the design and use of PNA molecules.

A "composition comprising a given polynucleotide sequence" as usedherein refers broadly to any composition containing the givenpolynucleotide sequence. The composition may comprise a dry formulationor an aqueous solution. Compositions comprising polynucleotide sequencesencoding NHETP (SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5) or fragmentsthereof (e.g., SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6 and fragmentsthereof) may be employed as hybridization probes. The probes may bestored in freeze-dried form and may be associated with a stabilizingagent such as a carbohydrate. In hybridizations, the probe may bedeployed in an aqueous solution containing salts (e.g., NaCl),detergents (e.g., SDS) and other components (e.g., Denhardt's solution,dry milk, salmon sperm DNA, etc.). "Consensus", as used herein, refersto a nucleic acid sequence which has been resequenced to resolveuncalled bases, has been extended using the XL-PCR kit (Perkin Elmer,Norwalk, Conn.) in the 5' and/or the 3' direction and resequenced, orhas been assembled from the overlapping sequences of more than oneIncyte Clone using a computer program for fragment assembly (e.g.,GELVIEW Fragment Assembly system, GCG, Madison, Wis.). Some sequenceshave been both extended and assembled to produce the consensus sequence.

The term "correlates with expression of a polynucleotide", as usedherein, indicates that the detection of the presence of ribonucleic acidthat is similar to SEQ ID 0:2, SEQ ID 20 NO:4, or SEQ ID NO:6 bynorthern analysis is indicative of the presence of m-RNA encoding NHETPin a sample and thereby correlates with expression of the transcriptfrom the polynucleotide encoding the protein.

A "deletion", as used herein, refers to a change in the amino acid ornucleotide sequence and results in the absence of one or more amino acidresidues or nucleotides. The term "derivative", as used herein, refersto the chemical modification of a nucleic acid encoding or complementaryto NHETP or the encoded NHETP. Such modifications include, for example,replacement of hydrogen by an alkyl, acyl, or amino group. A nucleicacid derivative encodes a polypeptide which retains the biological orimmunological function of the natural molecule. A derivative polypeptideis one which is modified by glycosylation, pegylation, or any similarprocess which retains the biological or immunological function of thepolypeptide from which it was derived.

The term "homology", as used herein, refers to a degree ofcomplementarty. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence that at leastpartially inhibits an identical sequence from hybridizing to a targetnucleic acid is referred to using the functional term "substantiallyhomologous." The inhibition of hybridization of the completelycomplementary sequence to the target sequence may be examined using ahybridization assay (Southern or northern blot, solution hybridizationand the like) under conditions of low stringency. A substantiallyhomologous sequence or hybridization probe will compete for and inhibitthe binding of a completely homologous sequence to the target sequenceunder conditions of low stringency. This is not to say that conditionsof low stringency are such that non-specific binding is permitted; lowstringency conditions require that the binding of two sequences to oneanother be a specific (i.e., selective) interaction. The absence ofnon-specific binding may be tested by the use of a second targetsequence which lacks even a partial degree of complementarity (e.g.,less than about 30% identity). In the absence of non-specific binding,the probe will not hybridize to the second non-complementary targetsequence.

Human artificial chromosomes (HACs) are linear microchromosomes whichmay contain DNA sequences of 10K to 10M in size and contain all of theelements required for stable mitotic chromosome segregation andmaintenance (Harrington, J. J. et al. (1997) Nat Genet. 15:345-355).

The term "humanized antibody", as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

The term "hybridization", as used herein, refers to any process by whicha strand of nucleic acid binds with a complementary strand through basepairing.

The term "hybridization complex", as used herein, refers to a complexformed between two nucleic acid sequences by virtue of the formation ofhydrogen bonds between complementary G and C bases and betweencomplementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀ t or R₀ tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,paper, membranes, filters, chips, pins or glass slides, or any otherappropriate substrate to which cells or their nucleic acids have beenfixed).

An "insertion" or "addition", as used herein, refers to a change in anamino acid or nucleotide sequence resulting in the addition of one ormore amino acid residues or nucleotides, respectively, as compared tothe naturally occurring molecule. "Microarray" refers to an array ofdistinct polynucleotides or oligonucleotides synthesized on a substrate,such as paper, nylon or other type of membrane, filter, chip, glassslide, or any other suitable solid support.

The term "modulate", as used herein, refers to a change in the activityof NHETP. For example, modulation may cause an increase or a decrease inprotein activity, binding characteristics, or any other biological,functional or immunological properties of NHETP.

"Nucleic acid sequence" as used herein refers to an oligonucleotide,nucleotide, or polynucleotide, and fragments thereof, and to DNA or RNAof genomic or synthetic origin which may be single- or double-stranded,and represent the sense or antisense strand. "Fragments" are thosenucleic acid sequences which are greater than 60 nucleotides than inlength, and most preferably includes fragments that are at least 100nucleotides or at least 1000 nucleotides, and at least 10,000nucleotides in length.

The term "oligonucleotide" refers to a nucleic acid sequence of at leastabout 6 nucleotides to about 60 nucleotides, preferably about 15 to 30nucleotides, and more preferably about 20 to 25 nucleotides, which canbe used in PCR amplification or a hybridization assay, or a microarray.As used herein, oligonucleotide is substantially equivalent to the terms"amplimers","primers", "oligomers", and "probes", as commonly defined inthe art.

"Peptide nucleic acid", PNA as used herein, refers to an antisensemolecule or anti-gene agent which comprises an oligonucleotide of atleast five nucleotides in length linked to a peptide backbone of aminoacid residues which ends in lysine. The terminal lysine conferssolubility to the composition. PNAs may be pegylated to extend theirlifespan in the cell where they preferentially bind complementary singlestranded DNA and RNA and stop transcript elongation (Nielsen, P. E. etal. (1993) Anticancer Drug Des. 8:53-63).

The term "portion", as used herein, with regard to a protein (as in "aportion of a given protein") refers to fragments of that protein. Thefragments may range in size from five amino acid residues to the entireamino acid sequence minus one amino acid. Thus, for example, a protein"comprising at least a portion of the amino acid sequence of SEQ IDNO:1" encompasses the full-length NHETP-1 and fragments thereof.

The term "sample", as used herein, is used in its broadest sense. Abiological sample suspected of containing nucleic acid encoding NHETP,or fragments thereof, or NHETP itself may comprise a bodily fluid,extract from a cell, chromosome, organelle, or membrane isolated from acell, a cell, genomic DNA, RNA, or cDNA(in solution or bound to a solidsupport, a tissue, a tissue print, and the like.

The terms "specific binding" or "specifically binding", as used herein,refers to that interaction between a protein or peptide and an agonist,an antibody and an antagonist. The interaction is dependent upon thepresence of a particular structure (i.e., the antigenic determinant orepitope) of the protein recognized by the binding molecule. For example,if an antibody is specific for epitope "A", the presence of a proteincontaining epitope A (or free, unlabeled A) in a reaction containinglabeled "A" and the antibody will reduce the amount of labeled A boundto the antibody.

The terms "stringent conditions" or "stringency", as used herein, referto the conditions for hybridization as defined by the nucleic acid,salt, and temperature. These conditions are well known in the art andmay be altered in order to identify or detect identical or relatedpolynucleotide sequences. Numerous equivalent conditions comprisingeither low or high stringency depend on factors such as the length andnature of the sequence (DNA, RNA, base composition), nature of thetarget (DNA, RNA, base composition, milieu (in solution or immobilizedon a solid substrate), concentration of salts and other components(e.g., formamide, dextran sulfate and/or polyethylene glycol), andtemperature of the reactions (within a range from about 5° C. below themelting temperature of the probe to about 20° C. to 25° C. below themelting temperature). One or more factors be may be varied to generateconditions of either low or high stringency different from, butequivalent to, the above listed conditions.

The term "substantially purified", as used herein, refers to nucleic oramino acid sequences that are removed from their natural environment,isolated or separated, and are at least 60% free, preferably 75% free,and most preferably 90% free from other components with which they arenaturally associated.

A "substitution", as used herein, refers to the replacement of one ormore amino acids or nucleotides by different amino acids or nucleotides,respectively.

"Transformation", as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the type of host cell beingtransformed and may include, but is not limited to, viral infection,electroporation, heat shock, lipofection, and particle bombardment. Such"transformed" cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

A "variant" of NHETP, as used herein, refers to an amino acid sequencethat is altered by one or more amino acids. The variant may have"conservative" changes, wherein a substituted amino acid has similarstructural or chemical properties, e.g., replacement of leucine withisoleucine. More rarely, a variant may have "nonconservative" changes,e.g., replacement of a glycine with a tryptophan. Analogous minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software.

THE INVENTION

The invention is based on the discovery of a new human electrontransport proteins (hereinafter referred to collectively as "NHETP", andindividually as NHETP-1, NHETP-2, and NHETP-3 ), the polynucleotidesencoding NHETP, and the use of these compositions for the diagnosis,prevention, or treatment of cancer, immune disorders, and reproductivedisorders.

Nucleic acids encoding the NHETP-1 of the present invention were firstidentified in Incyte Clone 1709102 from the prostate tissue cDNA library(PROSNOT16) using a computer search for amino acid sequence alignments.A consensus sequence, SEQ ID NO:2, was derived from the followingoverlapping and/or extended nucleic acid sequences: Incyte Clones 166518(LIVRNOT01), 529762 (BRAINOT03), 1397948 (BRAITUT08), 1709102(PROSNOT16), and 2636170 (BONTNOT01).

Nucleic acids encoding the NHETP-2 of the present invention were firstidentified in Incyte Clone 2235994 from the pancreatic tumor cDNAlibrary (PANCTUT02) using a computer search for amino acid sequencealignments. A consensus sequence. SEQ ID NO:4, was derived from thefollowing overlapping and/or extended nucleic acid sequences: IncyteClones 125647 (LUNGNOT01) and 2235994 (PANCTUT02).

Nucleic acids encoding the NHETP-3 of the present invention were firstidentified in Incyte Clone 2378038 from the pancreatic islet cell cDNAlibrary (ISLTNOT01) using a computer search for amino acid sequencealignments. A consensus sequence, SEQ ID NO:6, was derived from thefollowing overlapping and/or extended nucleic acid sequences: IncyteClones 1995822 (BRSTTUT03), 2378038 (ISLTNOT01), and 2598914(UTRSNOT10).

In one embodiment, the invention encompasses a polypeptide comprisingthe amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1A, 1B, 1C,and 1D. NHETP-1 is 305 amino acids in length and has a potentialN-linked glycosylation site at residue N₂₁₄. Numerous potential proteinkinase phosphorylation sites are found for casein kinase II at T₃₉, T₈₇,T₁₀₂, S₁₀₃, S₁₆₉, and T₂₁₆, and for protein kinase C at S₃₃, T₆₁, S₈₆,S₁₃₇, T₁₅₆, T₂₁₆, and S₂₉₈. As shown in FIG. 4, NHETP-1 has chemical andstructural homology with cytochrome b5 reductase from cow, b5R(GI162941; SEQ ID NO:7). In particular, NHETP-1 and bovine b5R share 65%identity. The N-linked glycosylation site at N₂₁₄ is shared by b5R. Mostof the potential protein kinase phosphorylation sites found in NHETP-1are also found in b5R, e.g., residues T₃₄, S₉₇, S₉₈, T₂₁₁, T₅₆, S₈₁,S₁₃₂, and T₂₁₁ The active site lysine residues identified in b5R at K₄₁,K₁₂₅, and K₁₆₃ are found in NHETP-1. As illustrated by FIGS. 7A and 7B,NHETP-1 and b5R have rather similar hydrophobicity plots. In particular,the amphipathic nature of both molecules, characteristic of cytochromeb5 reductase, is evident from these figures in terms of numeroushydrophobic membrane-anchoring segments adjacent to hydrophilic segmentsthat may extend into the aqueous environment of the cell. Northernanalysis shows the expression of this sequence in various libraries, atleast 4(6% of which are immortalized or cancerous, at least 23% of whichinvolve inflammation and the immune response, and at least 24% of whichinvolve the reproductive system and fetal development. Of particularnote is the expression of NHETP-1 in various inflammatory conditionsincluding ulcerative colitis, rheumatoid arthritis, lymphocyticthyroiditis, asthma, and Gaucher's disease.

In another embodiment, the invention encompasses a polypeptidecomprising the amino acid sequence of SEQ ID NO:3, as shown in FIGS. 2Aand 2B. NHETP-2 is 171 amino acids in length and has a potentialmitochondrial import signal peptide extending from approximatelyresidues M₁ to G₂₀. It is characterized by the presence of numeroushydrophobic residues, including alanine, glycine, and valine, and basicresidues lysine and arginine. An arginine residue at position -2relative to the potential cleavage site at G₂₀ is also present. NHETP-2contains a potential N-linked glycosylation site at N₈₉ and potentialprotein kinase phosphorylation sites for casein kinase II at T₇₆, T₉₁,T₁₃₄, and S₁₆₀, and for protein kinase C at T₂₇. A potentialmyristoylation site is also found at G₂₀ which would become theN-terminal residue of the mature protein following processing of thesignal peptide. As shown in FIG. 5, NHETP-2 has chemical and structuralhomology with human cytochrome c oxidase subunit 4, COX4 (GI 180935; SEQID NO:8). In particular, NHETP-2 and COX4 share 50% homology. COX4 alsocontains a mitochondrial import signal peptide extending fromapproximately residues M1 to A23. The sequence of this signal peptidediffers significantly from that of NHETP-2. The remaining portions ofthe two molecules, the mature proteins, show a much higher degree ofhomology. Three of the four potential casein kinase II phosphorylationsites in NHETP-2 are found in COX4 at residues S₇₄, S₈₉ and S₁₅₈, Asillustrated by FIGS. 8A and 8B, NHETP-2 and COX4 have rather similarhydrophobicity plots. In particular, a rather prominent region ofhydrophobicity is evident in both proteins, centered at approximatelyresidue 100. Northern analysis shows the expression of this sequence invarious tissues, at least 50% of which are immortalized or cancerous,and at least 13% of which involve inflammation and the immune response.

In another embodiment, the invention encompasses a polypeptidecomprising the mino acid sequence of SEQ ID NO:5, as shown in FIGS. 3Aand 3B. NHETP-3 is 128 amino acids in length and has potential proteinkinase C phosphorylation sites at T₅₅ and T₉₃. As shown in FIG. 6,NHETP-3 has chemical and structural homology with NADH dehydrogenasesubunit B14 from cow (GI 240; SEQ ID NO:9). In particular, NHETP-3 andbovine subunit B14 share 90% identity. Both protein kinase Cphosphorylation sites found in NHETP-3 are found in bovine B14. Asillustrated in FIGS. 9A and 9B, NHETP-3 and bovine NADH-D subunit B14have rather similar hydrophobicity plots. Northern analysis shows theexpression of this sequence in various tissues, at least 43% of whichare immortalized or cancerous, at least 14% of which involveinflammation and the immune response, and at least 28% involve thereproductive system. Of particular note is the expression of NHETP-3 incancers of the reproductive system including breast, testicles,prostate, and uterus.

The invention also encompasses NHETP variants. A preferred NHETP variantis one having at least 80%, and more preferably at least 90%, amino acidsequence icentity to the NHETP amino acid sequence (SEQ ID NO:1, SEQ IDNO:3, or SEQ ID NO:5) and which retains at least one biological,immunological, or other functional characteristic or activity of NHETP.A most preferred NHETP variant is one having at least 95% amino acidsequence identity to SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5.

The invention also encompasses polynucleotides which encode NHETP.Accordingly, any nucleic acid sequence which encodes the amino acidsequence of NHETP can be used to produce recombinant molecules whichexpress NHETP. In a particular embodiment, the invention encompasses thepolynucleotide comprising the nucleic acid sequence of SEQ ID NO:2 asshown in FIGS. 1A, 1B, 1C, and 1D. In another embodiment, the inventionencompasses the polynucleotide comprising the nucleic acid sequence ofSEQ ID NO:4 as shown in FIGS. 2A and 2B. In still another embodiment,the invention encompasses the polynucleotide comprising the nucleic acidsequence of SEQ ID NO:6 as shown in FIGS. 3A and 3B.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of nucleotide sequencesencoding NHETP, some bearing minimal homology to the nucleotidesequences of any known and naturally occurring gene, may be produced.Thus, the invention contemplates each and every possible variation ofnucleotide sequence that could be made by selecting combinations basedon possible codon choices. These combinations are made in accordancewith the standard triplet genetic code as applied to the nucleotidesequence of naturally occurring NHETP, and all such variations are to beconsidered as being specifically disclosed.

Although nucleotide sequences which encode NHETP and its variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring NHETP under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding NHETP or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding NHETP and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

The invention also encompasses production of DNA sequences, or fragmentsthereof, which encode NHETP and its derivatives, entirely by syntheticchemistry. After production, the synthetic sequence may be inserted intoany of the many available expression vectors and cell systems usingreagents that are well known in the art. Moreover, synthetic chemistrymay be used to introduce mutations into a sequence encoding NHETP or anyfragment thereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed nucleotide sequences, and inparticular, those shown in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6,under various conditions of stringency as taught in Wahl, G. M. and S.L. Berger (1987; Methods Enzymol. 152:399-407) and Kimmel, A. R. (1987;Methods Enzymol. 152:507-511).

Methods for DNA sequencing which are well known and generally availablein the art and may be used to practice any of the embodiments of theinvention. The methods may employ such enzymes as the Klenow fragment ofDNA polymerase I, SEQUENASE (US Biochemical Corp, Cleveland, Ohio.), Taqpolymerase (Perkin Elmer), thermostable T7 polymerase (Amersham,Chicago, IL.), or combinations of polymerases and proofreadingexonucleases such as those found in the ELONGASE amplification system(GIBCO/BRL, Gaithersburg, Md.). Preferably, the process is automatedwith machines such as the Hamilton MICROLAB 2200 (Hamilton, Reno, Nev.),Peltier thermal cycler (PTC200; MJ Research, Watertown, Mass.) and theABI CATALYST and 373 and 377 DNA Sequencers (Perkin Elmer).

The nucleic acid sequences encoding NHETP may be extended utilizing apartial nucleotide sequence and employing various methods known in theart to detect upstream sequences such as promoters and regulatoryelements. For example, one method which may be employed,"restriction-site" PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322). In particular, genomic DNA is first amplified in thepresence of primer to a linker sequence and a primer specific to theknown region. The amplified sequences are then subjected to a secondround of PCR with the same linker primer and another specific primerinternal to the first one. Products of each round of PCR are transcribedwith an appropriate RNA polymerase and sequenced using reversetranscriptase.

Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, T. et al. (1988)Nucleic Acids Res. 16:8186). The primers may be designed usingcommercially available software such as OLIGO 4.06 primer analysissoftware (National Biosciences Inc., Plymouth, Minn.), or anotherappropriate program, to be 22-30 nucleotides in length, to have a GCcontent of 50% or more, and to anneal to the target sequence attemperatures about 68°-72° C. The method uses several restrictionenzymes to generate a suitable fragment in the known region of a gene.The fragment is then circularized by intramolecular ligation and used asa PCR template.

Another method which may be used is capture PCR which involves PCRamplification of DNA fragments adjacent to a known sequence in human andyeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCRMethods Applic. 1:111-119). In this method, multiple restriction enzymedigestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown fragment of the DNA moleculebefore performing PCR.

Another method which may be used to retrieve unknown sequences is thatof Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PromoterFinderTmlibraries to walk genomic DNA (Clontech, Palo Alto, Calif.). Thisprocess avoids the need to screen libraries and is useful in findingintronlexon junctions.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable, in that they will contain moresequences which contain the 5' regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into 5' non-transcribedregulatory regions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentfluorescent dyes (one for each nucleotide) which are laser activated,and detection of the emitted wavelengths by a charge coupled devisecamera. Output/light intensity may be converted to electrical signalusing appropriate software (e.g. GENOTYPE and SEQUENCE NAVIGATOR, PerkinElmer) and the entire process from loading of samples to computeranalysis and electronic data display may be computer controlled.Capillary electrophoresis is especially preferable for the sequencing ofsmall pieces of DNA which might be present in limited amounts in aparticular sample.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode NHETP may be used in recombinant DNAmolecules to direct expression of NHETP, fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced, and these sequences may be used to clone and expressNHETP.

As will be understood by those of skill in the art, it may beadvantageous to produce NHETP-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce an RNA transcript havingdesirable properties, such as a half-life which is longer than that of atranscript generated from the naturally occurring sequence.

The nucleotide sequences of the present invention can be engineeredusing methods generally known in the art in order to alter NHETPencoding sequences for a variety of reasons, including but not limitedto, alterations which modify the cloning, processing, and/or expressionof the gene product. DNA shuffling by random fragmentation and PCRreassembly of gene fragments and synthetic oligonucleotides may be usedto engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, introduce mutations, and so forth.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding NHETP may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of NHETP activity, it may be useful toencode a chimeric NHETP protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleaviage site located between the NHETP encoding sequence and theheterologous protein sequence, so that NHETP may be cleaved and purifiedaway from the heterologous moiety.

In another embodiment, sequences encoding NHETP may be synthesized, inwhole or in part, using chemical methods well known in the art (seeCaruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 7:215-223,Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 7:225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of NHETP, or a fragment thereof.For example, peptide synthesis can be performed using varioussolid-phase techniques (Roberge, J. Y. et al. (1995) Science269:202-204) and automated synthesis may be achieved, for example, usingthe ABI 431 A peptide synthesizer (Perkin Elmer).

The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins, Stractures and Molecular Principles, WH Freeman andCo., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton, supra). Additionally, the amino acidsequence of NHETP, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

In order to express a biologically active NHETP, the nucleotidesequences encoding NHETP or functional equivalents, may be inserted intoappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding NHETP andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

A variety of expression vector/host systems may be utilized to containand express sequences encoding NHETP. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Theinvention is not limited by the host cell employed.

The "control elements" or "regulatory sequences" are thosenon-translated regions of the vector-enhancers, promoters, 5' and 3'untranslated regions-which interact with host cellular proteins to carryout transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene,LaJolla, Calif.) or PSPORTI plasmid (Gibco BRL) and the like may beused. The baculovirus olyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding NHETP,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for NHETP. For example, when largequantities of NHETP are needed for the induction of antibodies, vectorswhich direct high level expression of fusion proteins that are readilypurified may be used. Such vectors include, but are not limited to, themultifunctional E. coli cloning and expression vectors such as theBLUESCRIPT phagermind (Stratagene), in which the sequence encoding NHETPmay be ligated into the vector in frame with sequences for theamino-terminal Met and the subsequent 7 residues of β-galactosidase sothat a hybrid protein is produced; pIN vectors (Van Heeke, G. and S. M.Schuster (1989) J. Biol. Chem. 264:5503-5509); and the like. PGEXvectors (Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel et al. (supra)and Grant et al. (1987) Methods Enzymol. 153:516-544.

In cases where plant expression vectors are used, the expression of,equences encoding NHETP may be driven by any of a number of promoters.For example, viral promoters such as the 35S and 19S promoters of CaMVmay be used alone or in combination with the omega leader sequence fromTMV (Takamatsu, N. (1987) EMBO J. 6:307-311). Alternatively, plantpromoters such as the small subunit of RUBISCO or heat shock promotersmay be used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R.et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) ResultsProbl. Cell Differ. 17:85-105). These constructs can be introduced intoplant cells by direct DNA transformation or pathogen-mediatedtransfection. Such techniques are described in a number of generallyavailable reviews (see, for example, Hobbs, S. or Murry, L. E. in McGrawHill Yearbook of Science and Technology (1992) McGraw Hill, New York,N.Y.; pp. 191-196.

An insect system may also be used to express NHETP. For example, in. onesuch system, Autographa californica nuclear polyhedrosis virus (AcNPV)is used as a vector to express foreign genes in Spodoptera frugiperdacells or in Trichoplusia larvae. The sequences encoding NHETP may becloned into a non-essential region of the virus, such as the polyhedringene, and placed under control of the polyhedrin promoter. Successfulinsertion of NHETP will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. frugiperda cells or Trichoplusialarvae in which NHETP may be expressed (Engelhard, E. K. et al. (1994)Proc. Nat. Acad. Sci. 91:3224-3227).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, sequences encoding NHETP may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain a viable virus which iscapable of expressing NHETP in infected host cells (Logan, J. and Shenk,T. (1984) Proc. NatI. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

Human artificial chromosomes (HACs) may also be employed to deliverlarger fragments of DNA than can be contained and expressed in aplasmid. HACs, of 6 to 10M are constructed and delivered viaconventional delivery methods (liposomes, polycationic amino polymers,or vesicles) for therapeutic purposes.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding NHETP. Such signals include the ATGinitiation codon and adjacent sequences. In cases where sequencesencoding NHETP, its initiation codon, and upstream sequences areinserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a fragment thereof, is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers which are appropriate for theparticular cell system which is used, such as those described in theliterature (Scharf, D. et al. (1994) Results Probl. Cell Differ.20:125-162).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a "prepro" form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells which have specific cellular machineryand characteristic mechanisms for post-translational activities (e.g.,CHO, HeLa, MVDCK, HEK293, and W138), are available from the AmericanType Culture Collection (ATCC; Bethesda, Md) and may be chosen to ensurethe correct modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressNHETP may be transformed using expression vectors which may containviral origins of replication and/or endogenous expression elements and aselectable marker gene on the same or on a separate vector. Followingthe introduction of the vector, cells may be allowed to grow for 1-2days in an enriched media before they are switched to selective media.The purpose of the selectable marker is to confer resistance toselection, and its presence allows growth and recovery of cells whichsuccessfully express the introduced sequences. Resistant clones ofstably transformed cells may be proliferated using tissue culturetechniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adeninephosphoribosyltransferase (Lowy, 1. et al. (1980) Cell 22:817-23) geneswhich can be employed in tk⁻ or aprt⁻ cells, respectively. Also,antimetabolite, antibiotic or herbicide resistance can be used as thebasis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.77:3567-70); npt, which confers resistance to the aminoglycoside,;neomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14) and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers, as anthocyanins, β glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression mayneed to be confirmed. For example, if the sequence encoding NHETP isinserted within a marker gene sequence, transformed cells containingsequences encoding NHETP can be identified by the absenece of markergene function. Alternatively, a marker gene can be placed in tandem witha sequence encoding NHETP under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

Alternatively, host cells which contain the nucleic acid sequenceencoding NHETP and express NHETP may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein.

The presence of polynucleotide sequences encoding NHETP can be detectedby DNA-DNA or DNA-RNA hybridization or amplification using probes orfragments or fragments of polynucleotides encoding NHETP. Nucleic acidamplification based assays involve the use of oligonucleotides oroligomers based on the sequences encoding NHETP to detect transformantscontaining DNA or RNA encoding NHETP.

A variety of protocols for detecting and measuring the expression ofNHETP, using either polyclonal or monoclonal antibodies specific for theprotein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson NUETP is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding NHETP includeoligolabeling, nick translation, end-labeling or PCR amplification usinga labeled nucleotide. Alternatively, the sequences encoding NHETP, orany fragments thereof may be cloned into a vector for the production ofan mRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize R NA probes in vitro byaddition of an appropriate RNA polymerase such as T7, T3, or SP6 andlabeled nucleotides. These procedures may be conducted using a varietyof commercially available kits (Pharmacia & Upjohn, (Kalamazoo, Mich.);Promega (Madison Wis.); and U.S. Biochemical Corp. (Cleveland, Ohio.).Suitable reporter molecules or labels, which may be used for ease ofdetection, include radionuclides, enzymes, fluorescent,chemiluminescent, or chromogenic agents as well as substrates,cofactors, inhibitors, magnetic particles, and the like.

Host cells transformed with nucleotide sequences encoding NHETP may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a transformedcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeNHETP may be designed to contain signal sequences which direct secretionof NHETP through a prokaryotic or eukaryotic cell membrane. Otherconstructions may be used to join sequences encoding NHETP to nucleotidesequence encoding a polypeptide domain which will facilitatepurification of soluble proteins. Such purification facilitating domainsinclude. but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAG extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen, San Diego, Calif.) between the purificationdomain and NHETP may be used to facilitate purification. One suchexpression vector provides for expression of a fusion protein containingNHETP and a nucleic acid encoding 6 histidine residues preceding athioredoxin or an enterokinase cleavage site. The histidine residuesfacilitate purification on IMIAC (immobilized metal ion affinitychromatography as described in Porath, J. et al. (1992, Prot. Exp.Purif. 3:263-281) while the enterokinase cleavage site provides a meansfor purifying NHETP from the fusion protein. A discussion of vectorswhich contain fusion proteins is provided in kroll, D. J. et al. (1993;DNA Cell Biol. 12:441-453).

In addition to recombinant production, fragments of NHETP may beproduced by direct peptide synthesis using solid-phase techniques(Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesismay be performed using manual techniques or by automation. Automatedsynthesis may be achieved, for example, using Applied Biosystems 431Apeptide synthesizer (Perkin Elmer). Various fragments of NHETP may bechemically synthesized separately and combined using chemical methods toproduce the fill length molecule.

THERAPEUTICS

Chemical and structural homology exists between NHETP-1 and cytochromeb5 reductase from cow (GI 162941). In addition, NHETP-1 is expressed incancer, tissues associated with inflammation and the immune response andwith the reproductive system.

Therefore, NHETP-1 appears to play a role in cancer, immune disorders,and reproductive disorders. In particular, increased expression oractivity of NHETP-1 appears to be associated with these conditions anddisorders.

In one embodiment, an antagonist of NHETP-1 may be administered to asubject to prevent or treat cancer. Types of cancer may include, but arenot limited to, adenocarcinoma, leukemia, lymphoma, melanoma, myeloma,sarcoma, and teratocarcinoma, and, in particular, cancers of the adrenalgland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus. In one aspect, an antibodywhich specifically binds NHETP-1 may be used directly as an antagonistor indirectly as a targeting or delivery mechanism for bringing apharmaceutical agent to cells or tissue which express NHETP-1.

In another embodiment, a vector expressing the complement of thepolynucleotide encoding NHETP-1 may be administered to a subject totreat or prevent a cancer including, but not limited to, the types ofcancer described above.

In another embodiment, an antagonist of NHETP-1 may be administered to asubject to prevent or treat an immune disorder. Such disorders mayinclude, but are not limited to, AIDS, Addison's disease, adultrespiratory distress syndrome, allergies, anemia, asthma,atherosclerosis, bronchitis, cholecystitis, Crohn's disease, ulcerativecolitis, atopic dermatitis, dermatomyositis, diabetes mellitus,emphysema, erythema nodosum, atrophic gastritis, glomerulonephritis,gout, Graves' disease, hypereosinophilia, irritable bowel syndrome,lupus erythematosus, multiple sclerosis, myasthenia gravis, myocardialor pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, andautoimmune thyroiditis; complications of cancer, hemodialysis,extracorporeal circulation; viral, bacterial, fungal, parasitic,protozoal, and helminthic infections, and trauma.

In another embodiment, a vector expressing the complement of thepolynucleotide encoding NHETP-1 may be administered to a subject totreat or prevent an immune disorder including, but not limited to, thosedescribed above.

In another embodiment, an antagonist of NHETP-1 may be administered to asubject to prevent or treat a reproductive disorder. Such disorders mayinclude, but are not limited to, disorders of prolactin production;infertility including tubal disease, ovulatory defects, andendometriosis; disruptions of the estrous cycle, disruptions of themenstrual cycle, polycystic ovary syndrome, ovarian hyperstimulationsyndrome, endometrial and ovarian tumors, autoimmune disorders, ectopicpregnancy, and teratogenesis; cancer of the breast, fibrocystic breastdisease, and galactorrhea; and disruptions of spermatogenesis, abnormalsperm physiology, cancer of the testis, cancer of the prostate, benignprostatic hyperplisia, prostatitis, carcinoma of the male breast, andgynecomastia.

In another embodiment, a vector expressing the complement of thepolynucleotide encoding NHETP-1 may be administered to a subject totreat or prevent a reproductive disorder including, but not limited to,those described above.

Chemical and structural homology exists between NHETP-2 and cytochrome coxidase subunit 4 from human (GI 180935). In addition, NHETP-2 isexpressed in cancer and in tissues associated with inflammation and theimmune response. Therefore, NHETP-2 appears to play a role in cancer andimmune disorders. In particular, increased expression or activity ofNHETP-2 appears to be associated with these conditions and disorders.

Therefore, in another embodiment, an antagonist of NHETP-2 may beadministered to a subject to prevent or treat cancer. Types of cancermay include, but are not limited to, adenocarcinoma, leukemia, lymphoma,melanoma, myeloma, sarcoma, and teritocarcinoma, and, in particular,cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast,cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney,liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate,salivary glands, skin, spleen, testis, thymus, thyroid, and uterus. Inone aspect, an antibody which specifically binds NHETP-2 may be useddirectly as an antagonist or indirectly as a targeting or deliverymechanism for bringing a pharmaceutical agent to cells or tissue whichexpress NHETP-2.

In another embodiment, a vector expressing the complement of thepolynucleotide encoding NHETP-2 may be administered to a subject totreat or prevent a cancer including, but not limited to, the types ofcancer described above.

In another embodiment, an antagonist of NHETP-2 may be administered to asubject to prevent or treat an immune disorder. Such disorders mayinclude, but are not limited to, AIDS, Addison's disease, adultrespiratory distress syndrome, allergies, anemia, asthma,atherosclerosis, bronchitis, cholecystitis, Crohn's disease, ulcerativecolitis, atopic dermatitis, dermatomyositis, diabetes mellitus,emphysema, erythema nodosum, atrophic: gastritis, glomerulonephritis,gout, Graves' disease, hypereosinophilia, irritable bowel syndrome,lupus erythematosus, multiple sclerosis, myasthenia gravis, myocardialor pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, rheumatoid arthritis, scleroderna, Sjogren's syndrome, andautoimmune thyroiditis; complications of cancer, hemodialysis,extracorporeal circulation; viral, bacterial, fungal, parasitic,protozoal, and helminthic infections, and trauma.

In another embodiment, a vector expressing the complement of thepolynucleotide encoding NHETP-2 may be administered to a subject totreat or prevent an immune disorder including, but not limited to, thosedescribed above.

Chemical and structural homology exists between NHETP-3 and NADHdehydrogenase subunit B14 from cow (GI 240). In addition, NHETP-3 isexpressed in cancer, and in tissues associated with inflammation and theimmune response and with the reproductive system. Therefore, NHETP-3appears to play a role in cancer, immune disorders, and reproductivedisorders. In particular, increased expression or activity of NHETP-3appears to be associated with these conditions and disorders.

In another embodiment, an antagonist of NHETP-3 may be administered to asubject to prevent or treat cancer. Types of cancer may include, but arenot limited to, adenocarcinoma, leukemia, lymphoma, melanoma, myeloma,sarcoma, and teratocarcinoma, and, in particular, cancers of the adrenalgland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thyinus, thyroid, and uterus. In one aspect, an antibodywhich specifically binds NHETP-3 may be used directly as an antagonistor indirectly as a targeting or delivery mechanism for bringing apharmaceutical agent to cells or tissue which express NHETP-3.

In another embodiment, a vector expressing the complement of thepolynucleotide encoding NHETP-3 may be administered to a subject totreat or prevent a cancer including, but not limited to, the types ofcancer described above.

In another embodiment, an antagonist of NHETP-3 may be administered to asubject to prevent or treat an immune disorder. Such disorders mayinclude, but are not limited to, AIDS, Addison's disease, adultrespiratory distress syndrome, allergies, anernia, asthma,atherosclerosis, bronchitis, cholecystitis, Crohn's disease, ulcerativecolitis, atopic dermatitis, dermatomyositis, diabetes mellitus,emphysema, erythema nodosum, atrophic gastritis, glomerulonephritis,gout, Graves' disease, hypereosinophilia, irritable bowel syndrome,lupus erythematosus, multiple sclerosis, myasthenia gravis, myocardialor pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, andautoimmune thyroiditis; complications of cancer, hemodialysis,extracorporeal circulation; viral, bacterial, fungal, parasitic,protozoal, and helminthic infections, and trauma.

In another embodiment, a vector expressing the complement of thepolynucleotide encoding NHETP-3 may be administered to a subject totreat or prevent an immune disorder including, but not limited to, thosedescribed above.

In another embodiment, an antagonist of NHETP-3 may be administered to asubject to prevent or treat a reproductive disorder. Such disorders mayinclude, but are not limited to, disorders of prolactin production;infertility including tubal disease, ovulatory defects, andendometriosis; and disruptions of the estrous cycle, disruptions of themenstrual cycle, polycystic ovary syndrome, ovarian hyperstimulationsyndrome, endometrial and ovarian tumors, autoinimune disorders, ectopicpregnancy, and teratogenesis; cancer of the breast, fibrocystic breastdisease, and galactorrhea; disruptions of spermatogenesis, abnormalsperm physiology, cancer of the testis, cancer of the prostate, benignprostatic hyperplasia, prostatitis, carcinoma of the male breast, andgynecomastia.

In another embodiment, a vector expressing the complement of thepolynucleotide encoding NHETP-3 may be administered to a subject totreat or prevent a reproductive disorder including, but not limited to,those described above.

In other embodiments, any of the proteins, antagonists, antibodies,agonists, complementary sequences or vectors of the invention may beadministered in combination with other appropriate therapeutic agents.Selection of the appropriate agents for use in combination therapy maybe made by one of ordinary skill in the art, according to conventionalpharmaceutical principles. The combination of therapeutic agents may actsynergistically to effect the treatment or prevention of the variousdisorders described above. Using this approach, one may be able toachieve therapeutic efficacy with lower dosages of each agent, thusreducing the potential for adverse side effects.

An antagonist of NHETP may be produced using methods which are generallyknown in the art. In particular, purified NHETP may be used to produceantibodies or to screen libraries of pharmaceutical agents to identifythose which specifically bind NHETP.

Antibodies to NHETP may be generated using methods that are well knownin the art. Such antibodies may include, but are not limited to,polyclonal, monoclonal, chimeric, single chain, Fab fragments, andfragments produced by a Fab expression library. Neutralizing antibodies,(i.e., those which inhibit dimer formation) are especially preferred fortherapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith NHETP or any fragment or oligopeptide thereof which has immunogenicproperties. Depending on the host species, various adjuvants may be usedto increase immunological response. Such adjuvants include, but are notlimited to, Freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

It is preferred that the oligopeptides, peptides, or fragments used toinduce antibodies to NHETP have an amino acid sequence consisting of atleast five amino acids and more preferably at least 10 amino acids. Itis also preferable that they are identical to a portion of the aminoacid sequence of the natural protein, and they may contain the entireamino acid sequence of a small, naturally occurring molecule. Shortstretches of NHETP amino acids may be fused with those of anotherprotein such as keyhole limpet hemocyanin and antibody produced againstthe chimeric molecule.

Monoclonal antibodies to NHETP may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. etal. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. et al. (1983) Proc.Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al. (1984) Mol. Cell Biol.62:109-120).

In addition, techniques developed for the production of "chimericantibodies", the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Natire312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceNHETP-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobulin libraries(Burton D. R. (1991) Proc. Natl. Acad. Sci. 88:11120-3).

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature(Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86: 3833-3837; Winter,G. et al. (1991) Nature 349:293-299).

Antibody fragments which contain specific binding sites for NHETP mayalso be generated. For example, such fragments include, but are notlimited to, the F(ab')2 fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab')2 fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse, W. D. et al. (1989) Science 254:1275-1281).

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between NHETP and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering NHETP epitopes is preferred, but a competitivebinding assay may also be employed (Maddox, supra).

In another embodiment of the invention, the polynucleotides encodingNHETP, or any fragment or complement thereof, may be used fortherapeutic purposes. In one aspect, the complement of thepolynucleotide encoding NHETP may be used in situations in which itwould be desirable to block the transcription of the mRNA. Inparticular, cells may be transformed with sequences complementary topolynucleotides encoding NHETP. Thus, complementary molecules orfragments may be used to modulate NHETP activity, or to achieveregulation of gene function. Such technology is now well known in theart, and sense or antisense oligonucleotides or larger fragments, can bedesigned from various locations along the coding or control regions ofsequences encoding NHETP.

Expression vectors derived from retro viruses, adenovirus, herpes orvaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct vectors which will express nucleic acid sequencewhich is complementary to the polynucleotides of the gene encodingNHETP. These techniques are described both in Sambrook et al. (supra)and in Ausubel et al. (supra).

Genes encoding NHETP can be turned off by transforming a cell or tissuewith expression vectors which express high levels of a polynucleotide orfragment thereof which encodes NHETP. Such constructs may be used tointroduce untranslatable ,ense or antisense sequences into a cell. Evenin the absence of integration into the DNA, such vectors may continue totranscribe RNA molecules until they are disabled by endogenousnucleases. Transient expression may last for a month or more with anon-replicating vector and even longer if appropriate replicationelements are part of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning complementary sequences or antisense molecules (DNA, RNA, orPNA) to the control, 5' or regulatory regions of the gene encoding NHETP(signal sequence, promoters, enhancers, and introns). Oligonucleotidesderived from the transcription initiation site, e.g., between positions-10 and +10 from the start site, are preferred. Similarly, inhibitioncan be achieved using "triple helix" base-pairing methodology. Triplehelix pairing is useful because it causes inhibition of the ability ofthe double helix to open sufficiently for the binding of polymerases,transcription factors, or regulatory molecules. Recent therapeuticadvances using triplex DNA have been described in the literature (Gee,J. E. et al. (1994) In: Huber, B. E. and B. I. Carr, Molecular andImmunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y.). Thecomplementary sequence or antisense molecule may also be designed toblock translation of mRNA by preventing the transcript from binding toribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding NHETP.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Complementary ribonucleic acid molecules and ribozymes of the inventionmay be prepared by any method known in the art for the synthesis ofnucleic acid molecules. These include techniques for chemicallysynthesizing oligonucleotides such as solid phase phosphoramiditechemical synthesis. Alternatively, RNA molecules may be generated by invitro and in vivo transcription of DNA sequences encoding NHETP. SuchDNA sequences may be incorporated into a wide variety of vectors withsuitable RNA polymcrase promoters such as T7 or SP6. Alternatively,these cDNA constructs that synthesize corrlplementary RNA constitutivelyor inducibly can be introduced into cell lines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5' and/or 3' ends of the moleculeor the use of phosphorothioate or 2' O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection, by liposome injections orpolycationic amino polymers (Goldman, C. K. et al. (1997) NatureBiotechnology 15:462-66; incorporated herein by reference) may beachieved using methods which are well known in the air.

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical composition, in conjunction with a pharmaceuticallyacceptable carrier, for any of the therapeutic effects discussed above.Such pharmaceutical compositions may consist of NHETP, antibodies toNHETP, mimetics, agonists, antagonists, or inhibitors of NHETP. Thecompositions may be administered alone or in combination with at leastone other agent, such as stabilizing compound, which may be administeredin any sterile, biocompatible pharmaceutical carrier, including, but notlimited to, saline, buffered saline, dextrose, and water. Thecompositions may be administered to a patient alone, or in combinationwith other agents, drugs or hormones.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration maLybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such a, Hanks' solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Non-lipid polycationicamino polymers may also be used for delivery. Optionally, the suspensionmay also contain suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at apH range of 4.5 to 5.5, that is combined with buffer prior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of NHETP, such labeling would includeamount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually mice, rabbits, dogs, or pigs. The animal modelmay also be used to determine the appropriate concentration range androute of administration. Such information can then be used to determineuseful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example NHETP or fragments thereof, antibodies of NHETP,agonists, antagonists or inhibitors of NHETP, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio between therapeutic and toxic effects is thetherapeutic index, and it can be expressed as the ratio, LD50/ED50.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotidcs than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

DIAGNOSTICS

In another embodiment, antibodies which specifically bind NHETP may beused for the diagnosis of conditions or diseases characterized byexpression of NHETP, or in assays to monitor patients being treated withNHETP, agonists, antagonists or inhibitors. The antibodies useful fordiagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for NHETP includemethods which utilize the antibody and a label to detect NHETP in humanbody fluids or extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by joining them, eithercovalently or non-covalently, with a reporter molecule. A wide varietyof reporter molecules which are known in the art may be used, several ofwhich are described above.

A variety of protocols including ELISA, RIA, and FACS for measuringNHETP are known in the art and provide a basis for diagnosing altered orabnormal levels of NHETP expression. Normal or standard values for NHETPexpression are established by combining body fluids or cell extractstaken from normal mammalian subjects, preferably human, with antibody toNHETP under conditions suitable for complex formation The amount ofstandard complex formation may be quantified by various methods, butpreferably by photometric, means. Quantities of NHETP expressed insubject, control and disease, samples from biopsied tissues are comparedwith the standard values. Deviation between standard and subject valuesestablishes the parameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encodingNHETP may be used for diagnostic purposes. The polynucleotides which maybe used include oligonucleotide sequences, complementary RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofNHETP may be correlated with disease. The diagnostic assay may be usedto distinguish between absence, presence, and excess expression ofNHETP, and to monitor regulation of NHETP levels during therapeuticintervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding NHETP or elosely related molecules, may be used to identifynucleic acid sequences which encode NHETP. The specificity of the probe,whether it is made from a highly specific region, e.g., 10 uniquenucleotides in the 5' regulatory region, or a less specific region,e.g., especially in the 3' coding region, and the stringency of thehybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding NHETP, alleles, or related sequences.

Probes may also be used for the detection of related sequences, andshould preferably contain at least 50% of the nucleotides from any ofthe NHETP encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and derived from the nucleotide sequence ofSEQ ID NO:2 or from genomic sequence including promoter, enhancerelements, and introns of the naturally occurring NHETP.

Means for producing specific hybridization probes for DNAs encodingNHETP include the cloning of nucleic acid sequences encoding NHETP orNHETP derivatives into vectors for the production of mRNA probes. Suchvectors are known in the art, commercially available, and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, radionuclides such as 32P or 35S, or enzymatic labels, such asalkaline phosphatase coupled to the probe via avidinrbiotin couplingsystems, and the like.

Polynucleotide sequences encoding NHETP may be used for the diagnosis ofconditions or disorders which are associated with expression of NHETP.Examples of such conditions or disorders include cancers such asadenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, andteratocarcinoma, and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus; immune disorders such asAIDS, Addison's disease, adult respiratory distress syndrome, allergies,anemia, asthma, atherosclerosis, bronchitis, cholecystitis, Crohn'sdisease, ulcerative colitis, atopic dermatitis, dermatomyositis,diabetes mellitus, emphysema, erythema nodosum, atrophic gastritis,glomerulonephritis, gout, Graves' disease, hypereosinophilia, irritablebowel syndrome, lupus erythematosus, multiple sclerosis, myastheniagravis, myocardial or pericardial inflammation, osteoarthritis,osteoporosis, pancreatitis, polymyosilis, rheumatoid arthritis,scleroderma, Sogren's syndrome, and autoimmune thyroiditis;complications of cancer, hemodialysis, extracorporeal circulation;viral, bacterial, fungal, parasitic, protozoal, and helminthicinfections and trauma; and reproductive disorders such as disorders ofprolactin production; infertility including tubal disease, ovulatorydefects, and endometriosis; disruptions of the estrous cycle,disruptions of the menstrual cycle, polycystic: ovary syndrome, ovarianhyperstimulation syndrome, endometrial and ovarian tumors, autoimmunedisorders, ectopic pregnancy, and teratogenesis; cancer of the breast,fibrocystic breast disease, and galactorrhea; and disruptions ofspermatogenesis, abnormal sperm physiology, cancer of the testis, cancerof the prostate, benign prostatic hyperplasia, prostatitis, carcinoma ofthe male breast, and gynecomastia. The polynucleotide sequences encodingNHETP may be used in Southern or northern analysis, dot blot, or othermembrane-based technologies; in PCR technologies; or in dipstick, pin,ELISA assays or microarrays utilizing fluids or tissues from patientbiopsies to detect altered NHETP expression. Such qualitative orquantitative methods are well known in the art.

In a particular aspect, the nucleotide sequences encoding NHETP may beuseful in assays that detect activation or induction of various cancers,particularly those mentioned above. The nucleotide sequences encodingNHETP may be labeled by standard methods, and added to a fluid or tissuesample from a patient under conditions suitable for the formation ofhybridization complexes. After a suitable incubation period, the sampleis washed and the signal is quantitated and compared with a standardvalue. If the amount of signal in the biopsied or extracted sample issignificantly altered from that of a comparable control sample, thenucleotide sequences have hybridized with nucleotide sequences in thesample, and the presence of altered levels of nucleotide sequencesencoding NHETP in the sample indicates the presence of the associateddisease. Such assays may also be used to evaluate the efficacy of aparticular therapeutic treatment regimen in animal studies, in clinicaltrials, or in monitoring the treatment of an individual patient.

In order to provide a basis for the diagnosis of disease associated withcxpression of NHETP, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, which encodes NHETP, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects withthose from an experiment where a known amount of a substantiallypurified polynucleotide is used. Standard values obtained from normalsamples may be compared with values obtained from samples from patientswho are symptomatic for disease. Deviation between standard and subjectvalues is used to establish the presence of disease.

Once disease is established and a treatment protocol is initiated,hybridization assays may be repeated on a regular basis to evaluatewhether the level of expression in the patient begins to approximatethat which is observed in the normal patient. The results obtained fromsuccessive assays may be used to show the efficacy of treatment over aperiod ranging from several days to months.

With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding NHETP may involve the use of PCR. Such oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably consist of two nucleotide sequences,one with sense orientation (5'→3') and another with antisense (3'←5'),employed under optimized conditions for identification of a specificgene or condition. The same two oligomers, nested sets of oligomers, oreven a degenerate pool of oligomers may be employed under less stringentconditions for detection and/or quantitation of closely related DNA orRNA sequences.

Methods which may also be used to quantitate the expression of NHETPinclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and standard curves onto which the experimentalresults are interpolated (Melby, P. C. et al. (1993) J. Immunol.Methods, 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem.212:229-236). The speed of quantitation of multiple samples may beaccelerated by running the assay in an ELISA format where the oligomerof interest is presented in various dilutions and a spectrophotometricor colorimetric response gives rapid quantitation.

In further embodiments, an oligonucleotide derived from any of thepolynucleotide sequences described herein may be used as a target in amicroarray. The microarray can be used to monitor the expression levelof large numbers of genes simultaneously (to produce a transcriptimage), and to identify genetic variants, mutations and polymorphisrms.This information will be useful in determining gene function,understanding the genetic basis of disease, diagnosing disease, and indeveloping and monitoring the activity of therapeutic agents (Heller, R.et al. (1997) Proc. Natl. Acad. Sci. 94:2150-55).

In one embodiment, the microarray is prepared and used according to themethods described in PCT application WO95/11995 (Chee et al.), Lockhart,D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena, M. et al.(1996; Proc. Natl. Acad. Sci. 93:10614-10619), all of which areincorporated herein in their entirety by reference.

The microarray is preferably composed of a large number of unique,single-stranded nucleic acid sequences, usually either syntheticantisense oligonucleotides or fragments of cDNAs, fixed to a solidsupport. The oligonucleotides are preferably about 6-60 nucleotides inlength, more preferably 15-30 nucleotides in length, and most preferablyabout 20-25 nucleotides in length. For a certain type of microarray, itmay be preferable to use oligonucleotides which are only 7-10nucleotides in length. The microarray may contain oligonucleotides whichcover the known 5', or 3', sequence, sequential oligonucleotides whichcover the full length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarrzty may be oligonucleotides that are specific to a geneor genes of interest in which at least a fragment of the sequence isknown or that are specific to one or more unidentified cDNA which arecommon to a particular cell type, developmental or disease state.

In order to produce oligonucleotides to a known sequence for amicroarray, the gene of interest is examined using a computer algorithmwhich starts at the 5' or more preferably at the 3' end of thenucleotide sequence. The algorithm identifies oligomers of definedlength that are unique to the gene, have a GC content within a rangesuitable for hybridization, and lack predicted secondary structure thatmay interfere with hybridization. In certain situations it may beappropriate to use pairs of oligonucleotides on a microarray. The"pairs" will be identical, except for one nucleotide which preferably islocated in the center of the sequence. The second oligonucleotide in thepair (mismatched by one) serves as a control. The number ofoligonucleotide pairs may range from two to one million. The oligomersare synthesized at designated areas on a substrate using alight-directed chemical process. The substrate may be paper, nylon orother type of membrane, filter, chip, glass slide or any other suitablesolid support.

In another aspect, an oligonucleotide may be synthesized on the surfaceof the substrate by using a chemical coupling procedure and an ink jetapplication apparatus, as described in PCT application WO95/251116(Baldeschweiler et al.) which is incorporated herein in its entirety byreference. In another aspect, a "gridded" array analogous to a dot (orslot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536 or 6144 oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

In order to conduct sample analysis using a microarray, the RNA or DNAfrom a biological sample is made into hybridization probes. The mRNA isisolated, and cDNA is produced and used as a template to make antisenseRNA (aRNA). The aRNA is amplified in the presence of fluorescentnucleotides, and labeled probes are incubated with the microarray sothat the probe sequences hybridize to complementary oligonucleotides ofthe microarray. Incubation conditions are adjusted so that hybridizationoccurs with precise complementary matches or with various degrees ofless complementarity. After removal of nonhybridized probes, a scanneris used to determine the levels and patterns of fluorescence. Thescanned images are examined to determine degree of complementarity andthe relative abundance of each oligonucleotide sequence on themicroarray. The biological samples may be obtained from any bodilyfluids (such as blood, urine, saliva, phlegm, gastric juices, etc.),cultured cells, biopsies, or other tissue preparations. A detectionsystem may be used to measure the absence, presence, and amount ofhybridization for all of the distinct sequences simultaneously. Thisdata may be used for large scale correlation studies on the sequences,mutations, variants, or polymorphisms among samples.

In another embodiment of the invention, the nucleic acid sequences whichencode NHETP may also be used to generate hybridization probes which areuseful for mapping the naturally occurring genomic sequence. Thesequences may be mapped to a particular chromosome, to a specific regionof a chromosome or to artificial chromosome constructions, such as humanartificial chromosomes (HACs), yeast artificial chromosomes (YACs),bacterial artificial chromosomes (BACs), bacterial P1 constructions orsingle chromosome cDNA libraries as reviewed in Price, C. M. (1993)Blood Rev. 7:127-134, and Trask, B. J. (1991) Trends Genet. 7:149-154.

Fluorescent in situ hybridization (FISH as described in Verma et al.(1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press,New York, N.Y.) may be correlated with other physical chromosome mappingtechniques and genetic map data.

Examples of genetic map data can be found in various scientific journalsor at Online Mendelian Inheritance in Man (OMIM). Correlation betweenthe location of the gene encoding NHETP on a physical chromosomal mapand a specific disease , or predisposition to a specific disease, mayhelp delimit the region of DNA associated with that genetic disease. Thenucleotide sequences of the subject invention may be used to detectdifferences in gene sequences between normal, carrier, or affectedindividuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques such as linkage analysis using established chromosomalmarkers may be used for extending genetic maps. Often the placement of agene on the chromosome of another mammalian species, such as mouse, mayreveal associated markers even if the number or arm of a particularhuman chromosome is not known. New sequences can be assigned tochromosomal arms, or parts thereof, by physical mapping. This providesvaluable information to investigators searching for disease genes usingpositional cloning or other gene discovery techniques. Once the diseaseor syndrome has been crudely localized by genetic linkage to aparticular genomic region, for example, AT to 11q22-23 (Gatti, R. A. etal. (1988) Nature 336:577-580), any sequences mapping to that area mayrepresent associated or regulatory genes for further investigation. Thenucleotide sequence of the subject invention may also be used to detectdifferences in the chromosomal location due to tramslocation, inversion,etc. among normal, carrier, or affected individuals.

In another embodiment of the invention, NHETP, its catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated. intracellularly. The formation of binding complexes, betweenNHETP and the agent being tested, may be measured.

Another technique for drug screening which may be used provides for highthroughput screening of compounds having suitable binding affinity tothe protein of interest as described in published PCT applicationWO84/03564. In this method, as; applied to NHETP large numbers ofdifferent small test compounds are synthesized on a solid substrate,such as plastic pins or some other surface. The test compounds arereacted with NHETP, or fragments thereof, and washed. Bound NHETP isthen detected by methods well known in the art. Purified NHETP can alsobe coated directly onto plates for use in the aforementioned drugscreening techniques. Alternatively, non-neutralizing antibodies can beused to capture the peptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding NHETP specificallycompete with a test compound for binding NHETP. In this manner, theantibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with NHETP.

In additional embodiments, the nucleotide sequences which encode NHETPmay be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

The examples below are provided to illustrate the subject invention andare not included for the purpose of limiting the invention.

EXAMPLES I cDNA Library Construction

PROSNOT16

The PROSTNOT 16 cDNA library was constructed from microscopically normalprostate obtained from a 68-year-old Caucasian male. The normal prostatetissue was excised during a radical prostatectomy along with prostatetissue for which the pathology report indicated was associated with aGleason grade 3+4 adenocarcinoma which perforated the capsule to involveperiprostatic tissue. Surgical margins (distal urethra, right and leftbladder bases, right and left apices) were negative for tumor.Initially, the patient presented with elevated prostate specific antigen(PSA), after which he was diagnosed with a malignant neoplasm of theprostate and myasthenia gravis. The patient history included benignhypertension, cerebrovascular disease, arteriosclerotic coronary arterydisease, osteoarthritis, type II diabetes without complications, acutemyocardial infarction, and alcohol use. The patient's family historyincluded benign hypertension, an episode of acute myocardial infarction,and hyperlipidemia in the patient's mother, and arterioscleroticcoronary artery disease and an episode of acute myocardial infarction inthe patient's sibling.

PANCTUT02

The PANCTUT02 cDNA library was constructed from cancerous pancreatictissue obtained from a 45-year old Caucasian female during a radicalpancreaticoduodenectomy. Pathology indicated a grade 4 anaplasticcarcinoma at the head of the pancreas. The tumor had infiltrated andulcerated the duodenal mucosa. The margins of resection, includingpancreas and common bile duct were free of involvement. A singlepericholedochal and a single peripancreatic lymph node were negative fortumor. Pathology also indicated chronic cholecystitis. The patientpresented with abdominal pain, nausea, vomiting and functional diarrhea.Patient history included tobacco use. Family history included benignhypertension and hyperlipidemia in mother and atherosclerosis in agrandparent.

ISLTNOT01

The ISLTNOT01 cDNA library was constructed from total RNA isolated frommicroscopically normal pancreatic islet cells (specimen #A143, Pfizer,Inc., New York, N.Y.). The mRNA was then isolated using the OLIGOTEX kit(QIAGEN, Inc.; Chatsworth, Calif.) and used to construct the cDNAlibrary.

The frozen tissue was homogenized and lysed using a Brinkmann POLYTRONNOMOGENIZER PT-3000 (Brinkmann Instruments, Westbury, N.J.) inguanidinium isothiocyanate solution. The lysates were centrifuged over a5.7 M CsCl cushion using an EBeckman SW28 rotor in a Beckman L8-70MUltracentrifuge (Beckman Instruments) for 18 hours at 25,000 rpm atambient temperature. RNA was extracted with acid phenol pH 4.7,precipitated using 0.3 M sodium acetate and 2.5 volumes of ethanol,resuspended in RNAse-free water, and DNase treated at 37° C. RNAextraction and precipitatation were repeated as before. The mRNA wasthen isolated using the OLIGOTEX kit (QIAGEN, Inc., Chatsworth, Calif.)and used to construct the cDNA libraries.

The mRNAs were handled according to the recommended protocols in theSUPERSCRIPT plasmid system (Cat. #18248-013, Gibco/BRL, Gaithersburg,Md.). cDNAs were fractionated on a SEPHAROSE CL4B column (Cat.#275105-01, Pharmacia), and those cDNAs exceeding 400 bp were ligatedinto pINCY. The plasmid pINCY was subsequently transformed into DH5° C.competent cells (Cat. #18258-012, Gibco/BRL).

II Isolation and Sequencing of cDNA Clones

Plasmid DNA was released from the cells and purified using the R.E.A.L.PREP 96 plasmid Kit (Catalog #26173, QIAGEN, Inc.). This kit enabled thesimultaneous purification of 96 samples in a 96-well block usingmulti-channel reagent dispensers. The recommended protocol was employedexcept for the following changes: 1) the bacteria were cultured in 1 mlof sterile Terrific Broth (Catalog #22711, Gibco/BRL) with carbenicillinat 25 mg/L and glycerol at 0.4%; 2) after inoculation, the cultures wereincubated for 19 hours and at the end of incubation, the cells werelysed with 0.3 ml of lysis buffer; and 3) following isopropanolprecipitation, the plasmid DNA pellet was resuspended in 0.1 ml ofdistilled water. After the last step in the protocol, samples weretransferred to a 96-well block for storage at 4° C.

The cDNAs were sequenced according to the method of Sanger et al. (1975,J. Mol. Biol. 94:441f), using a Hamilton MICRO LAB 2200 (Hamilton, Reno,Nev.) in combination with Peltier thermal cyclers (PTC200 from MJResearch, Watertown, Mass.) and Applied Biosystems 377 DNA sequencingsystems; and the reading frame was determined.

III Homology Searching of cDNA Clones and Their Deduced Proteins

The nucleotide sequences and/or amino acid sequences of the SequenceListing were used to query sequences in the GenBank, SwissProt, BLOCKS,and Pima II databases. These databases, which contain previouslyidentified and annotated sequences, were searched for regions ofhomology using BLAST, which stands for Basic Local Alignment Search Tool(Altschul, S. F. (1993) J. Mol. Evol 36:290-300; Altschul, et al. (1990)J. Mol. Biol. 215:403-410).

BLAST produced alignments of both nucleotide and amino acid sequences todetermine sequence similarity. Because of the local nature of thealignments, BLAST was especially useful in determining exact matches orin identifying homologs which may be of prokaryotic (bacterial) oreukaryotic (animal, fungal, or plant) origin. Other algorithms such asthe one described in Smith, T. et al. (1992, Protein Engineering5:35-51), incorporated herein by reference, could have been used whendealing with primary sequence patterns and secondary structure gappenalties. The sequences disclosed in this application have lengths ofat least 49 nucleotides, and no more than 12% uncalled bases (where N isrecorded rather than A, C, G, or T).

The BLAST approach searched for matches between a query sequence and adatabase sequence. BLAST evaluated the statistical significance of anymatches found, and reported only those matches that satisfy theuser-selected threshold of significance. In this application, thresholdwas set at 10⁻²⁵ for nucleotides and 10⁻¹⁴ for peptides.

Incyte nucleotide sequences were searched against the GenBank databasesfor primate (pri), rodent (rod), and other mammalian sequences (mam);and deduced amino acid sequences from the same clones were then searchedagainst GenBank functional protein databases, mammalian (mamp),vertebrate (vrtp), and eukaryote (eukp) for homology.

IV Northern Analysis

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound (Sambrook et al., supra).

Analogous computer techniques using BLAST (Altschul, S. F. (1993)J.Mol.Evol. 36:290-300; Altschul, S. F. et al. (1990) J.Mol.Evol.215:403-410) are used to search for identical or related molecules innucleotide databases such as GenBank or the LIFESEQ™ database (IncytePharmaceuticals). This analysis is much faster than multiple,membranebased hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or homologous.

The basis of the search is the product score which is defined as:##EQU1## The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1-2% error; and at 70, the match will be exact. Homologous moleculesare usually identified by selecting those which show product scoresbetween 15 and 40, although lower scores may identify related molecules.

The results of northern analysis are reported as a list of libraries inwhich the transcript encoding NHETP occurs. Abundance and percentabundance are also reported. Abundance directly reflects the number oftimes a particular transcript is represented in a cDNA library, andpercent abundance is abundance divided by the total number of sequencesexamined in the cDNA library.

V Extension of NHETP Encoding Polynucleotides

The nucleic acid sequence of the Incyte Clone 1709102, 2235994, or2378038 was used to design oligonucleotide primers for extending apartial nucleotide sequence to full length. One primer was synthesizedto initiate extension in the antisense direction, and the other wassynthesized to extend sequence in the sense direction. Primers were usedto facilitate the extension of the known sequence "outward" generatingamplicons containing new, unknown nucleotide sequence for the region ofinterest. The initial primers were designed from the cDNA using OLIGO4.06 primer analysis software (National Biosciences), or anotherappropriate program, to be about 22 to about 30 nucleotides in length,to have a GC content of 50% or more, and to anneal to the targetsequence at temperatures of about 68° to about 72° C. Any stretch ofnucleotides which would result in hairpin structures and primer-primnerdimerizations was avoided.

Selected human cDNA libraries (Gibco/BRL) were used to extend thesequence If more than one extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

High fidelity amplification was obtained by following the instructionsfor the XL-PCR kit (Perkin Elmer) and thoroughly mixing the enzyme andreaction mix. Beginning with 40 pmol of each primer and the recommendedconcentrations of all other components of the kit, PCR was performedusing the Peltier thermal cycler (PTC200; M. J. Research, Watertown,Mass.) and the following parameters:

    ______________________________________    Step 1      94° C. for 1 min (initial denaturation)    Step 2               65° C. for 1 min    Step 3            68° C. for 6 min    Step 4             94° C. for 15 sec    Step 5               65° C. for 1 min    Step 6            68° C. for 7 min    Step 7               Repeat step 4-6 for 15 additional cycles    Step 8               94° C. for 15 sec    Step 9               65° C. for 1 min    Step 10             68° C. for 7:l5 min    Step 11             Repeat step 8-10 for 12 cycles    Step 12              72° C. for 8 min    Step 13            4° C. (and holding)    ______________________________________

A 5-10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gelto determine which reactions were successful in extending the sequence.Bands thought to contain the largest products were excised from the gel,purified using QIAQuick™ (QIAGEN Inc., Chatswortti, Calif.), and trimmedof overhangs using Klenow enzyme to facilitate religation and cloning.

After ethanol precipitation, the products were redissolved in 13 μl ofligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase were added, and the mixture was incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) were transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook et al., supra).After incubation for one hour at 37° C., the E. coli mixture was platedon Luria Bertani (LB)-agar (Sambrook et al., supra) containing 2x Carb.The following day, several colonies were randomly picked from each plateand cultured in 150 μl of liquid LB/2x Carb medium placed in anindividual well of an appropriate, commercially-available, sterile96-well microtiter plate. The following day, 5 μl of each overnightculture was transferred into a non-sterile 96-well plate and afterdilution 1:10 with water, 5 μl of each sample was transferred into a PCRarray.

For PCR amplification, 18 μl of concentrated PCR reaction mix (3.3x)containing 4 units of rTth DNA polymerase, a vector primer, and one orboth of the gene s;pecific primers used for the extension reaction wereadded to each well. Amplification was performed using the followingconditions:

    ______________________________________    Step 1     94° C. for 60 sec    Step 2               94° C. for 20 sec    Step 3               55° C. for 30 sec    Step 4              72° C. for 90 sec    Step 5              Repeat steps 2-4 for an additional 29 cycles    Step 6               72° C. for 180 sec    Step 7              4° C. (and holding)    ______________________________________

Aliquots of the PCR reactions were run on agarose gels together withmolecular weight markers. The sizes of the PCR products were compared tothe original partial cDNAs, and appropriate clones were selected,ligated into plasmid, and sequenced.

In like manner, the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:4, orSEQ ID NO:6 is used to obtain 5' regulatory sequences using theprocedure above, oligonucleotides designed for 5' extension, and anappropriate genomic library.

VI Labeling and Use of Individual Hybridization Probes

Hybridization probes derived from SEQ ID NO:2, SEQ ID NO:4, or SEQ IDNO:6 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although thelabeling of oligonucleotides, consisting of about 20 base-pairs, isspecifically described, essentially the same procedure is used withlarger nucleotide fragments. Oligonucleotides are designed usingstate-of-the-art software such as OLIGO 4.06 primer analysis software(National Biosciences), labeled by combining 50 pmol of each oligomerand 250 μCi of γ-³² P! adenosine triphosphate (Amersham) and T4polynucleotide kinase (DuPont NEN®, Boston, Mass.). The labeledoligonucleotides are substantially purified with SEPHADEX G-25 superfineresin column (Pharmacia & Upjohn). A aliquot containing 10⁷ counts perminute of the labeled probe is used in a typical membrane-basedhybridization analysis of human genomic DNA digested with one of thefollowing endonucleases (Ase I, Bgl II, Eco RI, Pst I, Xba 1, or Pvu II;DuPont NEN®).

The DNA from each digest is fractionated on a 0.7 percent agarose ge,land transferred to nylon membranes (NYTRAN PLUS, Schleicher & Schuell,Durham, NH). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1 x salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT ARAutoradiography film (Kodak, Rochester, N.Y.) is exposed to the blots ina PHOSPOIMAGER cassette (Molecular Dynamics, Sunnyvale, Calif.) forseveral hours, hybridization patterns are compared visually.

VII Microarrays

To produce oligonucleotides for a microarray, at least one of thenucleotide sequences described herein is examined using a computeralgorithm which starts at the 3' end of the nucleotide sequence. Thealgorithm identifies oligomers of defined length that are unique to thegene, have a GC content within a range suitable for hybridization, andlack predicted secondary structure that would interfere withhybridization. The algorithm identifies 20 sequence-specificoligonucleotides of 20 nucleotides in length (20-mers). A matched set ofoligonucleotides is created in which one nucleotide in the center ofeach sequence is altered. This process is repeated for each gene in themicroarray, and double sets of twenty 20 mers are synthesized andarranged on the surface of the silicon chip using a light-directedchemical process (Chee, M. et al., PCT/WO95/11995, incorporated hereinby reference).

In the alternative, a chemical coupling procedure and an ink jet deviceare used to synthesize oligomers on the surface of a substrate(Baldeschweiler, J. D. et al., PCT/WO95/25116, incorporated herein byreference). In another alternative, a "gridded" array analogous to a dot(or slot) blot is used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array may beproduced by hand or using available materials and machines and containgrids of 8 dots, 24 dots, 96 dots, 384 dots, 1536 dots, or 6144 dots.After hybridization, the microarray is washed to remove nonhybridizedprobes, and a scanner is used to determine the levels and patterns offluorescence. The scanned images are examined to determine degree ofcomplementarity and the relative abundance of each oligonucleotidesequence on the micro-array.

VIII Complementary Polynucleotides

Sequence complementary to the NHETP-encoding sequence, or any partthereof, is used to decrease or inhibit expression of naturallyoccurring NHETP. Although use of oligonucleotides comprising from about15 to about 30 base-pairs is described, essentially the same procedureis used with smaller or larger sequence fragments. Appropriateoligonucleotides are designed using Oligo 4.06 software and the codingsequence of NHETP, SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5. To inhibittranscription, a complementary oligonucleotide is designed from the mostunique 5' sequence and used to prevent promoter binding to the codingsequence. To inhibit translation, a complementary oligonucleotide isdesigned to prevent ribosomal binding to the NHETP-encoding transcript.

IX Expression of NHETP

Expression of NHETP is accomplished by subcloning the cDNAs intoappropriate vectors and transforming the vectors into host cells. Inthis case, the cloning vector is also used to express NHETP in E. coli.Upstream of the cloning site, this vector contains a promoter forγ-galactosidase, followed by sequence containing the amino-terminal Met,and the subsequent seven residues of γ-galactosidase. Immediatelyfollowing these eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

Induction of an isolated, transformed bacterial strain with IPTG usingstandard methods produces a fusion protein which consists of the firsteight residues of γ-galactosidase, about 5 to 15 residues of linker, andthe full length protein. The signal residues direct the secretion ofNHETP into the bacterial growth media which can be used directly in thefollowing assay for activity.

X Demonstration of NHETP Activity

NHETP-1

NHETP-1 activity is measured by the transfer of electrons from (andconsequent oxidation of) NADH to cytochrome b5 when NHETP-1 is incubatedtogether with NADH and cytochrome b5. The reaction is carried out in anoptical cuvette containing aliquots of NHETP-1 together with 150 μM eachof NADH and cytochrome b5 in 1 M Tris-acetate buffer, pH 8.1. Thereaction is incubated at 21° C. and the oxidation of NADH is followed bythe change in absorption at 340 nm using an ultravioletspectrophotometer. The activity of NHETP-1 is proportional to the rateof change of absorption at 340 nm.

NHETP-2

NHETP-2 activity is measured by the transfer of electrons fromcytochrome c to an electron acceptor (KCN) in the presence of areconstituted cytochrome c oxidase enzyme complex containing NHETP-2 inplace of COX4. The reconstituted cytochrome c oxidase is incubatedtogether with cytochrome c and KCN in a suitable buffer. The reaction iscarried out in an optical cuvette and monitored by the change inabsorption due to oxidation of cytochrome c using a spectrophotometer.Cytochrome c oxidase reconstituted in the absence of NHETP-2 is used asa negative control. The activity of NHETP-2 is proportional to thechange in optical absorption measured.

NHETP-3

NHETP-3 activity is measured in the reconstituted NADH-D complex by thecatalysis of electron transfer from NADH to decylubiquinone (DB). Thereaction contains 10 μg/mL NADH-D protein, 20 μM NADH in 50 mM tris-HCLbuffer, pH 7.5, 50 mM NaCl, and 1 mM KCN. The reaction is started byaddition of DB at 2 uM and followed by the change in absorbance at 340nm due to the oxidation of NADH using an ultraviolet spectrophotometer.NADH-D complex reconstituted in the absence of NHETP-3 is compared as anegative control. The activity of NHETP-3 in the reconstituted NADH-Dcomplex is proportional to the rate of change of absorbance at 340 nm.

XI Production of NHETP Specific Antibodies

NHETP that is substantially purified using PAGE electrophoresis(Sambrook, supra), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols. The aminoacid sequence deduced from SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 isanalyzed using DNASTAR software (DNASTAR Inc) to determine regions ofhigh immunogenicity and a corresponding oligopeptide is synthesized andused to raise antibodies by means known to those of skill in the art.Selection of approprliate epitopes, such as those near the C-terminus orin hydrophilic regions, is described by Ausubel et al. (supra), andothers.

Typically, the oligopeptides are 15 residues in length, synthesizedusing an Applied Biosystems 431A peptide synthesizer usingfmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH, Sigma,St. Louis, Mo.) by reaction withN-maleirmidobenzoyl-N-hydroxysuccinimide ester (MBS; Ausubel et al.,supra). Rabbits are immunized with the oligopeptide-KLH complex incomplete Freund's adjuvant. The resulting antisera are tested forantipeptide activity, for example, by binding the peptide to plastic,blocking with 1% BSA, reacting with rabbit antisera, washing, andreacting with radio iodinated, goat anti-rabbit IgG.

XII Purification of Naturally Occurring NHETP Using Specific Antibodies

Naturally occurring or recombinant NHETP is substantially purified byimmunoaffinity chromatography using antibodies specific for NHETP. Animmunoaffinity column is constructed by covalently coupling NHETPantibody to an activated chromatographic resin, such as CNBr-activatedSepharose (Pharmacia & Upjohn). After the coupling, the resin is blockedand washed according to the manufacturer's instructions.

Media containing NHETP is passed over the immunoaffinity column, and thecolumn is washed under conditions that allow the preferential absorbanceof NHETP (e.g., high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/NHETP binding (eg, a buffer of pH 2-3 or a high concentrationof a chaotrope, such as urea or thiocyanate ion), and NHETP iscollected.

XIII Identification of Molecules Which Interact with NHETP

NHETP or biologically active fragments thereof are labeled with ¹²⁵ IBolton-Hunter reagent (Bolton et al. (1973) Biochem. J. 133: 529).Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled NHETP, washed and any wells withlabeled NHETP complex are assayed. Data obtained using differentconcentrations of NHETP are used to calculate values for the number,affinity, and association of NHETP with the candidate molecules.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 9    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 305 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -    (vii) IMMEDIATE SOURCE:              (A) LIBRARY: PROSNOT16              (B) CLONE: 1709102    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    - Met Gly Ile Gln Thr Ser Pro Val Leu Leu Al - #a Ser Leu Gly Val Gly    #                15    - Leu Val Thr Leu Leu Gly Leu Ala Val Gly Se - #r Tyr Leu Val Arg Arg    #            30    - Ser Arg Arg Pro Gln Val Thr Leu Leu Asp Pr - #o Asn Glu Lys Tyr Leu    #        45    - Leu Arg Leu Leu Asp Lys Thr Thr Val Ser Hi - #s Asn Thr Lys Arg Phe    #    60    - Arg Phe Ala Leu Pro Thr Ala His His Thr Le - #u Gly Leu Pro Val Gly    #80    - Lys His Ile Tyr Leu Ser Thr Arg Ile Asp Gl - #y Ser Leu Val Ile Arg    #                95    - Pro Tyr Thr Pro Val Thr Ser Asp Glu Asp Gl - #n Gly Tyr Val Asp Leu    #           110    - Val Ile Lys Val Tyr Leu Lys Gly Val His Pr - #o Lys Phe Pro Glu Gly    #       125    - Gly Lys Met Ser Gln Tyr Leu Asp Ser Leu Ly - #s Val Gly Asp Val Val    #   140    - Glu Phe Arg Gly Pro Ser Gly Leu Leu Thr Ty - #r Thr Gly Lys Gly His    145                 1 - #50                 1 - #55                 1 -    #60    - Phe Asn Ile Gln Pro Asn Lys Lys Ser Pro Pr - #o Glu Pro Arg Val Ala    #               175    - Lys Lys Leu Gly Met Ile Ala Gly Gly Thr Gl - #y Ile Thr Pro Met Leu    #           190    - Gln Leu Ile Arg Ala Ile Leu Lys Val Pro Gl - #u Asp Pro Thr Gln Cys    #       205    - Phe Leu Leu Phe Ala Asn Gln Thr Glu Lys As - #p Ile Ile Leu Arg Glu    #   220    - Asp Leu Glu Glu Leu Gln Ala Arg Tyr Pro As - #n Arg Phe Lys Leu Trp    225                 2 - #30                 2 - #35                 2 -    #40    - Phe Thr Leu Asp His Pro Pro Lys Asp Trp Al - #a Tyr Ser Lys Gly Phe    #               255    - Val Thr Ala Asp Met Ile Arg Glu His Leu Pr - #o Ala Pro Gly Asp Asp    #           270    - Val Leu Val Leu Leu Cys Gly Pro Pro Pro Me - #t Val Gln Leu Ala Cys    #       285    - His Pro Asn Leu Asp Lys Leu Gly Tyr Ser Gl - #n Lys Met Arg Phe Thr    #   300    - Tyr    305    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 1617 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -    (vii) IMMEDIATE SOURCE:              (A) LIBRARY: PROSNOT16              (B) CLONE: 1709102    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    - GTCGGCTTGT CAGGTGGTGG AGGAAAAGGC GCTCCGTCAT GGGGATCCAG AC - #GAGCCCCG      60    - TCCTGCTGGC CTCCCTGGGG GTGGGGCTGG TCACTCTGCT CGGCCTGGCT GT - #GGGCTCCT     120    - ACTTGGTTCG GAGGTCCCGC CGGCCTCAGG TCACTCTCCT GGACCCCAAT GA - #AAAGTACC     180    - TGCTACGACT GCTAGACAAG ACGACTGTGA GCCACAACAC CAAGAGGTTC CG - #CTTTGCCC     240    - TGCCCACCGC CCACCACACT CTGGGGCTGC CTGTGGGCAA ACATATCTAC CT - #CTCCACCC     300    - GAATTGATGG CAGCCTGGTC ATCAGGCCAT ACACTCCTGT CACCAGTGAT GA - #GGATCAAG     360    - GCTATGTGGA TCTTGTCATC AAGGTCTACC TGAAGGGTGT GCACCCCAAA TT - #TCCTGAGG     420    - GAGGGAAGAT GTCTCAGTAC CTGGATAGCC TGAAGGTTGG GGATGTGGTG GA - #GTTTCGGG     480    - GGCCAAGCGG GTTGCTCACT TACACTGGAA AAGGGCATTT TAACATTCAG CC - #CAACAAGA     540    - AATCTCCACC AGAACCCCGA GTGGCGAAGA AACTGGGAAT GATTGCCGGC GG - #GACAGGAA     600    - TCACCCCAAT GCTACAGCTG ATCCGGGCCA TCCTGAAAGT CCCTGAAGAT CC - #AACCCAGT     660    - GCTTTCTGCT TTTTGCCAAC CAGACAGAAA AGGATATCAT CTTGCGGGAG GA - #CTTAGAGG     720    - AACTGCAGGC CCGCTATCCC AATCGCTTTA AGCTCTGGTT CACTCTGGAT CA - #TCCCCCAA     780    - AAGATTGGGC CTACAGCAAG GGCTTTGTGA CTGCCGACAT GATCCGGGAA CA - #CCTGCCCG     840    - CTCCAGGGGA TGATGTGCTG GTACTGCTTT GTGGGCCACC CCCAATGGTG CA - #GCTGGCCT     900    - GCCATCCCAA CTTGGACAAA CTGGGCTACT CACAAAAGAT GCGATTCACC TA - #CTGAGCAT     960    - CCTCCAGCTT CCCTGGTGCT GTTCGCTGCA GTTGTTCCCC ATCAGTACTC AA - #GCACTATA    1020    - AGCCTTAGAT TCCTTTCCTC AGAGTTTCAG GTTTTTTCAG TTACATCTAG AG - #CTGAAATC    1080    - TGGATAGTAC CTGCAGGAAC AATATTCCTG TAGCCATGGA AGAGGGCCAA GG - #CTCAGTCA    1140    - CTCCTTGGAT GGCCTCCTAA ATCTCCCCGT GGCAACAGGT CCAGGAGAGG CC - #CATGGAGC    1200    - AGTCTCTTCC ATGGAGTAAG AAGGAAGGGA GCATGTACGC TTGGTCCAAG AT - #TGGCTAGT    1260    - TCCTTGATAG CATCTTACTC TCACCTTCTT TGTGTCTGTG ATGAAAGGAA CA - #GTCTGTGC    1320    - AATGGGTTTT ACTTAAACTT CACTGTTCAA CCTATGAGCA AATCTGTATG TG - #TGAGTATA    1380    - AGTTGAGCAT AGCATACTTC CAGAGGTGGT CTTATGGAGA TGGCAAGAAA GG - #AGGAAATG    1440    - ATTTCTTCAG ATCTCAAAGG AGTCTGAAAT ATCATATTTC TGTGTGTGTC TC - #TCTCAGCC    1500    - CCTGCCCAGG CTAGAGGGAA ACAGCTACTG ATAATCGAAA ACTGCTGTTT GT - #GGCAGGAA    1560    - CCCCTGGCTG TGCAAATAAA TGGGGCTGAG GCCCCTGTGT GATATTGAAA AA - #AAAAA    1617    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 171 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -    (vii) IMMEDIATE SOURCE:              (A) LIBRARY: PANCTUT02              (B) CLONE: 2235994    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    - Met Leu Pro Arg Ala Ala Trp Ser Leu Val Le - #u Arg Lys Gly Gly Gly    #                15    - Gly Arg Arg Gly Met His Ser Ser Glu Gly Th - #r Thr Arg Gly Gly Gly    #            30    - Lys Met Ser Pro Tyr Thr Asn Cys Tyr Ala Gl - #n Arg Tyr Tyr Pro Met    #        45    - Pro Glu Glu Pro Phe Cys Thr Glu Leu Asn Al - #a Glu Glu Gln Ala Leu    #    60    - Lys Glu Lys Glu Lys Gly Ser Trp Thr Gln Le - #u Thr His Ala Glu Lys    #80    - Val Ala Leu Tyr Arg Leu Gln Phe Asn Glu Th - #r Phe Ala Glu Met Asn    #                95    - Arg Arg Ser Asn Glu Trp Lys Thr Val Met Gl - #y Cys Val Phe Phe Phe    #           110    - Ile Gly Phe Ala Ala Leu Val Ile Trp Trp Gl - #n Arg Val Tyr Val Phe    #       125    - Pro Pro Lys Pro Ile Thr Leu Thr Asp Glu Ar - #g Lys Ala Gln Gln Leu    #   140    - Gln Arg Met Leu Asp Met Lys Val Asn Pro Va - #l Gln Gly Leu Ala Ser    145                 1 - #50                 1 - #55                 1 -    #60    - His Trp Asp Tyr Glu Lys Lys Gln Trp Lys Ly - #s    #               170    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 658 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    - CGGACGGTGG TCCGCAGCGG GTTCTCATTG CTCGCTGGGC AGACCCAGGT CG - #CGCTCCCA      60    - CTGCCGAGCC CGCGAGATGC TCCCCAGAGC TGCCTGGAGC TTGGTGCTGA GG - #AAAGGTGG     120    - AGGTGGAAGA CGAGGGATGC ACAGCTCAGA AGGCACCACC CGTGGTGGGG GG - #AAGATGTC     180    - CCCCTACACC AACTGCTATG CCCAGCGCTA CTACCCCATG CCAGAAGAGC CC - #TTCTGCAC     240    - AGAACTCAAC GCTGAGGAGC AGGCCCTGAA GGAGAAGGAG AAGGGAAGCT GG - #ACCCAGCT     300    - GACCCACGCC GAAAAGGTGG CCTTGTACCG GCTCCAGTTC AATGAGACCT TT - #GCGGAGAT     360    - GAACCGTCGC TCCAATGAGT GGAAGACAGT GATGGGTTGT GTCTTCTTCT TC - #ATTGGATT     420    - CGCAGCTCTG GTGATTTGGT GGCAGCGGGT CTACGTATTT CCTCCAAAGC CG - #ATCACCTT     480    - GACGGACGAG CGGAAAGCCC AGCAGCTGCA GCGCATGCTG GACATGAAGG TG - #AATCCTGT     540    - GCAGGGCCTG GCCTCCCACT GGGACTATGA GAAGAAGCAG TGGAAGAAGT GA - #CTTGCATC     600    - CCCAGCTGTC TCCCTGAGGC TCCGCCCTGG CTGGGAGCCT CTGGCGGCCC CT - #CCCCTC     658    - (2) INFORMATION FOR SEQ ID NO:5:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 128 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -    (vii) IMMEDIATE SOURCE:              (A) LIBRARY: ISLTNOT01              (B) CLONE: 2378038    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    - Met Ala Gly Ser Gly Val Arg Gln Val Thr Se - #r Thr Ala Ser Thr Phe    #                15    - Val Lys Pro Ile Phe Ser Arg Asp Met Asn Gl - #u Ala Lys Arg Arg Val    #            30    - Arg Glu Leu Tyr Arg Ala Trp Tyr Arg Glu Va - #l Pro Asn Thr Val His    #        45    - Gln Phe Gln Leu Asp Ile Thr Val Lys Met Gl - #y Arg Asp Lys Val Arg    #    60    - Glu Met Phe Met Lys Asn Ala His Val Thr As - #p Pro Arg Val Val Asp    #80    - Leu Leu Val Ile Lys Gly Lys Ile Glu Leu Gl - #u Glu Thr Ile Lys Val    #                95    - Trp Lys Gln Arg Thr His Val Met Arg Phe Ph - #e His Glu Thr Glu Ala    #           110    - Pro Arg Pro Lys Asp Phe Leu Ser Lys Phe Ty - #r Val Gly His Asp Pro    #       125    - (2) INFORMATION FOR SEQ ID NO:6:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 506 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -    (vii) IMMEDIATE SOURCE:              (A) LIBRARY: ISLTNOT01              (B) CLONE: 2378038    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    - GCACGCGCTG CTTGCAAANG GGTGGGGTTG TGGAGTGGAT GCTTTGGCAA GA - #TGGCGGGG      60    - AGCGGCGTCC GCCAAGTTAC TTCTACCGCC AGCACCTTCG TGAAGCCCAT TT - #TCAGTCGG     120    - GACATGAACG AGGCCAAGCG GAGGGTGCGC GAGCTCTACC GCGCCTGGTA TC - #GGGAGGTG     180    - CCGAACACTG TGCACCAATT CCAGCTGGAC ATCACTGTGA AAATGGGACG GG - #ATAAAGTC     240    - CGAGAAATGT TTATGAAGAA TGCCCATGTC ACAGACCCCA GGGTGGTTGA TC - #TTCTGGTC     300    - ATTAAGGGAA AGATCGAACT GGAAGAAACA ATTAAAGTAT GGAAGCAGCG GA - #CACATGTT     360    - ATGCGGTTCT TCCATGAAAC AGAAGCGCCA AGGCCAAAGG ATTTCCTATC CA - #AGTTCTAT     420    - GTTGGCCACG ATCCATGAAG TCATTCAGTG GAAAGATGCA CGTTGATACT AT - #TTTAGAGC     480    #             506  TACA ATGGTC    - (2) INFORMATION FOR SEQ ID NO:7:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 300 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -    (vii) IMMEDIATE SOURCE:              (A) LIBRARY: GenBank              (B) CLONE: 162941    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    - Gly Ala Gln Leu Ser Thr Leu Gly His Val Va - #l Leu Ser Pro Val Trp    #                15    - Phe Leu Tyr Ser Leu Ile Met Lys Leu Phe Gl - #n Arg Ser Thr Pro Ala    #            30    - Ile Thr Leu Glu Asn Pro Asp Ile Lys Tyr Pr - #o Leu Arg Leu Ile Asp    #        45    - Lys Glu Val Ile Ser His Asp Thr Arg Arg Ph - #e Arg Phe Ala Leu Pro    #    60    - Ser Pro Glu His Ile Leu Gly Leu Pro Val Gl - #y Gln His Ile Tyr Leu    #80    - Ser Ala Arg Ile Asp Gly Asn Leu Val Ile Ar - #g Pro Tyr Thr Pro Val    #                95    - Ser Ser Asp Asp Asp Lys Gly Phe Val Asp Le - #u Val Ile Lys Val Tyr    #           110    - Phe Lys Asp Thr His Pro Lys Phe Pro Ala Gl - #y Gly Lys Met Ser Gln    #       125    - Tyr Leu Glu Ser Met Lys Ile Gly Asp Thr Il - #e Glu Phe Arg Gly Pro    #   140    - Asn Gly Leu Leu Val Tyr Gln Gly Lys Gly Ly - #s Phe Ala Ile Arg Pro    145                 1 - #50                 1 - #55                 1 -    #60    - Asp Lys Lys Ser Asp Pro Val Ile Lys Thr Va - #l Lys Ser Val Gly Met    #               175    - Ile Ala Gly Gly Thr Gly Ile Thr Pro Met Le - #u Gln Val Ile Arg Ala    #           190    - Ile Met Lys Asp Pro Asp Asp His Thr Val Cy - #s His Leu Leu Phe Ala    #       205    - Asn Gln Thr Glu Lys Asp Ile Leu Leu Arg Pr - #o Glu Leu Glu Glu Leu    #   220    - Arg Asn Glu His Ser Ala Arg Phe Lys Leu Tr - #p Tyr Thr Val Asp Lys    225                 2 - #30                 2 - #35                 2 -    #40    - Ala Pro Glu Ala Trp Asp Tyr Ser Gln Gly Ph - #e Val Asn Glu Glu Met    #               255    - Ile Arg Asp His Leu Pro Pro Pro Glu Glu Gl - #u Pro Leu Val Leu Met    #           270    - Cys Gly Pro Pro Pro Met Ile Gln Tyr Ala Cy - #s Leu Pro Asn Leu Asp    #       285    - Arg Val Gly His Pro Lys Glu Arg Cys Phe Al - #a Phe    #   300    - (2) INFORMATION FOR SEQ ID NO:8:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 169 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -    (vii) IMMEDIATE SOURCE:              (A) LIBRARY: GenBank              (B) CLONE: 180935    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    - Met Leu Ala Thr Arg Val Phe Ser Leu Val Gl - #y Lys Arg Ala Ile Ser    #                15    - Thr Ser Val Cys Val Arg Ala His Glu Ser Va - #l Val Lys Ser Glu Asp    #            30    - Phe Ser Leu Pro Ala Tyr Met Asp Arg Arg As - #p His Pro Leu Pro Glu    #        45    - Val Ala His Val Lys His Leu Ser Ala Ser Gl - #n Lys Ala Leu Lys Glu    #    60    - Lys Glu Lys Ala Ser Trp Ser Ser Leu Ser Me - #t Asp Glu Lys Val Glu    #80    - Leu Tyr Arg Ile Lys Phe Lys Glu Ser Phe Al - #a Glu Met Asn Arg Gly    #                95    - Ser Asn Glu Trp Lys Thr Val Val Gly Gly Al - #a Met Phe Phe Ile Gly    #           110    - Phe Thr Ala Leu Val Ile Met Trp Gln Lys Hi - #s Tyr Val Tyr Gly Pro    #       125    - Leu Pro Gln Ser Phe Asp Lys Glu Trp Val Al - #a Lys Gln Thr Lys Arg    #   140    - Met Leu Asp Met Lys Val Asn Pro Ile Gln Gl - #y Leu Ala Ser Lys Trp    145                 1 - #50                 1 - #55                 1 -    #60    - Asp Tyr Glu Lys Asn Glu Trp Lys Lys                    165    - (2) INFORMATION FOR SEQ ID NO:9:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 128 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -    (vii) IMMEDIATE SOURCE:              (A) LIBRARY: GenBank              (B) CLONE: 240    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    - Met Ala Ala Ser Gly Leu Arg Gln Ala Ala Va - #l Ala Ala Ser Thr Ser    #                15    - Val Lys Pro Ile Phe Ser Arg Asp Met Asn Gl - #u Ala Lys Arg Arg Val    #            30    - Arg Glu Leu Tyr Arg Ala Trp Tyr Arg Glu Va - #l Pro Asn Thr Val His    #        45    - Leu Phe Gln Leu Asp Ile Ser Val Lys Gln Gl - #y Arg Asp Lys Val Arg    #    60    - Glu Met Phe Lys Lys Asn Ala His Ile Thr As - #p Pro Arg Val Val Asp    #80    - Leu Leu Val Ile Lys Gly Lys Met Glu Leu Gl - #u Glu Thr Ile Lys Val    #                95    - Trp Lys Gln Arg Thr His Val Met Arg Phe Ph - #e His Glu Thr Glu Ala    #           110    - Pro Arg Pro Lys Asp Phe Leu Ser Lys Phe Ty - #r Val Gly His Asp Pro    #       125    __________________________________________________________________________

What is claimed is:
 1. An isolated and purified polynucleotide encodinga polypeptide comprising the amino acid sequence of SEQ ID NO:1.
 2. Acomposition comprising the polynucleotide of claim
 1. 3. An isolated andpurified polynucleotide which is completely complementary to thepolynucleotide of claim
 1. 4. An isolated and purified polynucleotidecomprising SEQ ID NO:2.
 5. An isolated and purified polynucleotide whichis completely complementary to the polynucleotide of claim
 4. 6. Anexpression vector containing the polynucleotide of claim
 1. 7. A hostcell containing the expression vector of claim
 6. 8. A method forproducing a polypeptide comprising the amino acid sequence of SEQ IDNO:1, the method comprising the steps of:a) culturing the host cell ofclaim 7 under conditions suitable for the expression of the polypeptide;and b) recovering the polypeptide from the host cell culture.